Methods, reagents and cells for biosynthesizing compounds

ABSTRACT

This document describes biochemical pathways for producing 6-hydroxyhexanoate methyl ester and hexanoic acid hexyl ester using one or more of a fatty acid O-methyltransferase, an alcohol O-acetyltransferase and a monooxygenase, as well as recombinant hosts expressing one or more of such enzymes. 6-hydroxyhexanoate methyl esters and hexanoic acid hexyl ester can be enzymatically converted to adipic acid, adipate semialdehyde, 6-aminohexanoate, 6-hydroxyhexanoate, hexamethylenediamine, and 1,6-hexanediol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.Nos. 62/012,659, filed Jun. 16, 2014, 62/012,666, filed Jun. 16, 2014,and 62/012,604, filed Jun. 16, 2014, the disclosure of each of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to methods for biosynthesizing 6-hydroxyhexanoatemethyl ester and hexanoic acid hexyl ester using one or more isolatedenzymes such as a fatty acid O-methyltransferase, an alcoholO-acetyltransferase and a monooxygenase, and to recombinant host cellsexpressing one or more such enzymes. This invention also relates tomethods for enzymatically converting 6-hydroxyhexanoate methyl ester andhexanoic acid hexyl ester to 6-hydroxyhexanoate and 1,6-hexanediol usingone or more enzymes such as an esterase, a monooxygenase and ademethylase. In addition, this invention relates to enzymaticallyconverting 6-hydroxyhexanoate and/or 1,6-hexanediol to adipic acid,6-aminohexanoic acid, hexamethylenediamine or 1,6-hexanediol (hereafter“C6 building blocks”), and recombinant hosts that produce such C6building blocks.

BACKGROUND

Nylons are polyamides which are generally synthesized by thecondensation polymerization of a diamine with a dicarboxylic acid.Similarly, Nylons may be produced by the condensation polymerization oflactams. A ubiquitous nylon is Nylon 6,6, which is produced bycondensation polymerization of hexamethylenediamine (HMD) and adipicacid. Nylon 6 can be produced by a ring opening polymerization ofcaprolactam (Anton & Baird, Polyamides Fibers, Encyclopedia of PolymerScience and Technology, 2001).

Biotechnology offers an alternative approach to petrochemical processesvia biocatalysis. Biocatalysis is the use of biological catalysts, suchas enzymes, to perform biochemical transformations of organic compounds.

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing one or more of adipic acid,6-hydroxyhexanoate, 6-aminohexanoate, hexamethylenediamine and1,6-hexanediol (hereafter “C6 building blocks”) wherein the methods arebiocatalyst based.

However, no wild-type prokaryote or eukaryote naturally overproduces orexcretes such C6 building blocks to the extracellular environment.Nevertheless, the metabolism of adipic acid has been reported.

The dicarboxylic acid adipic acid is converted efficiently as a carbonsource by a number of bacteria and yeasts via β-oxidation into centralmetabolites. β-oxidation of Coenzyme A (CoA) activated adipate to CoAactivated 3-oxoadipate facilitates further catabolism via, for example,pathways associated with aromatic substrate degradation. The catabolismof 3-oxoadipyl-CoA to acetyl-CoA and succinyl-CoA by several bacteriahas been characterized comprehensively

The optimality principle states that microorganisms regulate theirbiochemical networks to support maximum biomass growth. Beyond the needfor expressing heterologous pathways in a host organism, directingcarbon flux towards C6 building blocks that serve as carbon sourcesrather than as biomass growth constituents, contradicts the optimalityprinciple. For example, transferring the 1-butanol pathway fromClostridium species into other production strains has often fallen shortby an order of magnitude compared to the production performance ofnative producers (Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915).

The efficient synthesis of the seven carbon aliphatic backbone precursoris a key consideration in synthesizing one or more C6 building blocksprior to forming terminal functional groups, such as carboxyl, amine orhydroxyl groups, on the C6 aliphatic backbone.

SUMMARY

This document is based at least in part on the discovery that it ispossible to construct biochemical pathways for producing a seven carbonchain aliphatic backbone precursor in which one or two functionalgroups, i.e., carboxyl, amine, or hydroxyl, can be formed, leading tothe synthesis of one or more of adipic acid, 6-aminohexanoate,6-hydroxyhexanoate, hexamethylenediamine, and 1,6-hexanediol (hereafter“C6 building blocks). Adipic acid and adipate, 6-hydroxyhexanoic acidand 6-hydroxyhexanoate, and 6-aminohexanoic and 6-aminohexanoate areused interchangeably herein to refer to the relevant compound in any ofits neutral or ionized forms, including any salt forms thereof. It isunderstood by those skilled in the art that the specific form willdepend on pH.

One of skill in the art understands that compounds containing carboxylicacid groups (including, but not limited to, organic monoacids,hydroxyacids, aminoacids, and dicarboxylic acids) are formed orconverted to their ionic salt form when an acidic proton present in theparent compound either is replaced by a metal ion, e.g., an alkali metalion, an alkaline earth ion, or an aluminum ion; or coordinates with anorganic base. Acceptable organic bases include, but are not limited to,ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases include, butare not limited to, aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. A salt ofthe present invention is isolated as a salt or converted to the freeacid by reducing the pH to below the pKa, through addition of acid ortreatment with an acidic ion exchange resin.

One of skill in the art understands that compounds containing aminegroups (including, but not limited to, organic amines, aminoacids, anddiamines) are formed or converted to their ionic salt form, for example,by addition of an acidic proton to the amine to form the ammonium salt,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids including, but not limited to, acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like. Acceptable inorganicbases include, but are not limited to, aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, andthe like. A salt of the present invention is isolated as a salt orconverted to the free amine by raising the pH to above the pKb throughaddition of base or treatment with a basic ion exchange resin.

One of skill in the art understands that compounds containing both aminegroups and carboxylic acid groups (including, but not limited to,aminoacids) are formed or converted to their ionic salt form byeither 1) acid addition salts, formed with inorganic acids including,but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid, and the like; or formed with organic acidsincluding, but not limited to, acetic acid, propionic acid, hexanoicacid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lacticacid, malonic acid, succinic acid, malic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like. Acceptable inorganicbases include, but are not limited to, aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, andthe like, or 2) when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase. Acceptable organic bases include, but are not limited to,ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases include, butare not limited to, aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. A salt canof the present invention is isolated as a salt or converted to the freeacid by reducing the pH to below the pKa through addition of acid ortreatment with an acidic ion exchange resin.

Pathways, metabolic engineering and cultivation strategies describedherein rely on producing hexanoate methyl ester from hexanoate using,for example, a fatty acid O-methyltransferase and producing6-hydroxyhexanoate methyl ester from hexanoate methyl ester using, forexample, a monooxygenase. 6-hydroxyhexanoate can be produced from6-hydroxyhexanoate methyl ester using, for example, a demethylase or anesterase.

Pathways, metabolic engineering and cultivation strategies describedherein rely on producing hexanoic acid hexyl ester using, for example,an alcohol O-acetyltransferase and producing 6-hydroxyhexanoic acidhexyl ester, 6-hydroxyhexanoic acid 6-hydroxyhexyl ester and/or hexanoicacid 6-hydroxyhexyl ester from hexanoic acid hexyl ester using, forexample, a monooxygenase. 6-hydroxyhexanoate can be produced from6-hydroxyhexanoic acid hexyl ester and/or 6-hydroxyhexanoic acid6-hydroxyhexyl ester using, for example, an esterase. 1,6-hexanediol canbe produced from hexanoic acid 6-hydroxyhexyl ester and/or6-hydroxyhexanoic acid 6-hydroxyhexyl ester using, for example, anesterase.

CoA-dependent elongation enzymes or homologs associated with the carbonstorage pathways from polyhydroxyalkanoate accumulating bacteria areuseful for producing precursor molecules. See, e.g., FIGS. 1-2.

In the face of the optimality principle, the inventors discoveredsurprisingly that appropriate non-natural pathways, feedstocks, hostmicroorganisms, attenuation strategies to the host's biochemical networkand cultivation strategies may be combined to efficiently produce one ormore C6 building blocks.

In some embodiments, the C6 aliphatic backbone for conversion to a C6building block can be formed from acetyl-CoA via two cycles ofCoA-dependent carbon chain elongation using either NADH or NADPHdependent enzymes. See FIG. 1 and FIG. 2.

In some embodiments, an enzyme in the CoA-dependent carbon chainelongation pathway generating the C6 aliphatic backbone purposefullycontains irreversible enzymatic steps.

In some embodiments, the terminal carboxyl groups can be enzymaticallyformed using a thioesterase, an aldehyde dehydrogenase, a7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a5-oxopentanoate dehydrogenase or a monooxygenase. See FIG. 3 and FIG. 4.

In some embodiments, the terminal amine groups can be enzymaticallyformed using a ω-transaminase or a deacetylase. See FIG. 5 and FIG. 6.

In some embodiments, the terminal hydroxyl group can be enzymaticallyforming using a monooxygenase, an esterase or an alcohol dehydrogenase.See FIG. 3, FIG. 7 and FIG. 8. A monooxygenase (e.g., in combinationwith an oxidoreductase and/or ferredoxin) or an alcohol dehydrogenasecan enzymatically form a hydroxyl group. The monooxygenase can have atleast 70% sequence identity to any one of the amino acid sequences setforth in SEQ ID NOs: 13-15 or 27-28. An esterase can have at least 70%identity to the amino acid sequence set forth in SEQ ID NO: 26.

A ω-transaminase or a deacetylase can enzymatically form an amine group.The ω-transaminase can have at least 70% sequence identity to any one ofthe amino acid sequences set forth in SEQ ID NOs. 7-12.

A thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoatedehydrogenase, a 6-oxohexanoate dehydrogenase or a 5-oxopentanoatedehydrogenase can enzymatically form a terminal carboxyl group. Thethioesterase can have at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1.

A carboxylate reductase (e.g., in combination with a phosphopantetheinyltransferase) can form a terminal aldehyde group as an intermediate informing the product. The carboxylate reductase can have at least 70%sequence identity to any one of the amino acid sequences set forth inSEQ ID NOs. 2-6.

Any of the methods can be performed in a recombinant host byfermentation. The host can be subjected to a cultivation strategy underaerobic, anaerobic, or micro-aerobic cultivation conditions. The hostcan be cultured under conditions of nutrient limitation such asphosphate, oxygen or nitrogen limitation. The host can be retained usinga ceramic membrane to maintain a high cell density during fermentation.

In any of the methods, the host's tolerance to high concentrations of aC6 building block can be improved through continuous cultivation in aselective environment.

The principal carbon source fed to the fermentation can derive frombiological or non-biological feedstocks. In some embodiments, thebiological feedstock is, includes, or derives from, monosaccharides,disaccharides, lignocellulose, hemicellulose, cellulose, lignin,levulinic acid and formic acid, triglycerides, glycerol, fatty acids,agricultural waste, condensed distillers' solubles, or municipal waste.

In some embodiments, the non-biological feedstock is or derives fromnatural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatileresidue (NVR) or a caustic wash waste stream from cyclohexane oxidationprocesses, or a terephthalic acid/isophthalic acid mixture waste stream.

This document also features a recombinant host that includes at leastone exogenous nucleic acid encoding a fatty acid O-methyltransferase,and a monooxygenase, and produce 6-hydroxyhexanoate methyl ester. Such ahost further can include a demethylase or esterase and further produce6-hydroxyhexanoate. Such hosts further can include (i) a β-ketothiolaseor an acetyl-CoA carboxylase and a β-ketoacyl-[acp] synthase, (ii) a3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, (iii) anenoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoA reductase. The hostsalso further can include one or more of a thioesterase, an aldehydedehydrogenase, or a butanal dehydrogenase.

This document also features a recombinant host that includes at leastone exogenous nucleic acid encoding an alcohol O-acetyltransferase andproduce hexanoic acid hexyl ester. Such a host further can include amonooxygenase and an esterase and further produce 6-hydroxyhexanoateand/or 1,6-hexanediol. Such hosts further can include (i) aβ-ketothiolase or an acetyl-CoA carboxylase and a β-ketoacyl-[acp]synthase, (ii) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoAreductase, (iii) an enoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoAreductase. The hosts also further can include one or more of athioesterase, carboxylate reductase, an aldehyde dehydrogenase, abutanal or acetaldehyde dehydrogenase or an alcohol dehydrogenase.

A recombinant host producing 6-hydroxyhexanoate further can include oneor more of a monooxygenase, an alcohol dehydrogenase, an aldehydedehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxybutanoatedehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-oxobutanoatedehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7-oxoheptanoatedehydrogenase, the host further producing adipic acid or adipatesemialdehyde.

A recombinant host producing 6-hydroxyhexanoate further can include oneor more of a ω-transaminase, a 6-hydroxyhexanoate dehydrogenase, a5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase,and an alcohol dehydrogenase, wherein the host further produces6-aminohexanoate.

A recombinant host producing 6-aminohexanoic acid can include anamidohydrolase, wherein the host further produces caprolactam.

A recombinant host producing 6-hydroxyhexanoate or 6-aminohexanoatefurther can include one or more of a carboxylate reductase, aω-transaminase, a deacetylase, a N-acetyl transferase, or an alcoholdehydrogenase, the host further producing hexamethylenediamine.

A recombinant host producing 6-hydroxyhexanoate further can include acarboxylate reductase or an alcohol dehydrogenase, wherein the hostfurther produces 1,6-hexanediol.

The recombinant host can be a prokaryote, e.g., from the genusEscherichia such as Escherichia coli; from the genus Clostridia such asClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; from the genus Corynebacteria such as Corynebacteriumglutamicum; from the genus Cupriavidus such as Cupriavidus necator orCupriavidus metallidurans; from the genus Pseudomonas such asPseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans;from the genus Delftia acidovorans, from the genus Bacillus such asBacillus subtillis; from the genes Lactobacillus such as Lactobacillusdelbrueckii; from the genus Lactococcus such as Lactococcus lactis orfrom the genus Rhodococcus such as Rhodococcus equi.

The recombinant host can be a eukaryote, e.g., a eukaryote from thegenus Aspergillus such as Aspergillus niger; from the genusSaccharomyces such as Saccharomyces cerevisiae; from the genus Pichiasuch as Pichia pastoris; from the genus Yarrowia such as Yarrowialipolytica, from the genus Issatchenkia such as Issathenkia orientalis,from the genus Debaryomyces such as Debaryomyces hansenii, from thegenus Arxula such as Arxula adenoinivorans, or from the genusKluyveromyces such as Kluyveromyces lactis.

In some embodiments, the host's endogenous biochemical network isattenuated or augmented to (1) ensure the intracellular availability ofacetyl-CoA, (2) create a cofactor, i.e. NADH or NADPH, imbalance thatmay be balanced via the formation of a C6 Building Block, (3) preventdegradation of central metabolites, central precursors leading to andincluding C6 Building Blocks and (4) ensure efficient efflux from thecell.

Any of the recombinant hosts described herein further can include one ormore of the following attenuated enzymes: polyhydroxyalkanoate synthase,an acetyl-CoA thioesterase, an acetyl-CoA specific β-ketothiolase, aphosphotransacetylase forming acetate, an acetate kinase, a lactatedehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoaciddecarboxylase producing isobutanol, an alcohol dehydrogenase formingethanol, a triose phosphate isomerase, a pyruvate decarboxylase, aglucose-6-phosphate isomerase, a transhydrogenase dissipating thecofactor imbalance, aglutamate dehydrogenase specific for the co-factorfor which an imbalance is created, a NADH/NADPH-utilizing glutamatedehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenaseaccepting C6 building blocks and central precursors as substrates; aglutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.

Any of the recombinant hosts described herein further can overexpressone or more genes encoding: an acetyl-CoA synthetase, a6-phosphogluconate dehydrogenase; a transketolase; a feedback resistantthreonine deaminase; a puridine nucleotide transhydrogenase; a formatedehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; aglucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; apropionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamatedehydrogenase; a L-glutamine synthetase; a lysine transporter; adicarboxylate transporter; and/or a multidrug transporter.

This document also features methods of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester, the method including enzymaticallyconverting a C₄₋₉ carboxylic acid to a (C₃₋₈ alkyl)-C(═O)OCH₃ ester; andenzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

In some embodiments, the C₄₋₉ carboxylic acid can be enzymaticallyconverted to the (C₃₋₉ alkyl)-C(═O)OCH₃ ester using a polypeptide havingfatty acid O-methyltransferase activity. In some embodiments, thepolypeptide having fatty acid O-methyltransferase activity can have atleast 70% sequence identity to an amino acid sequence set forth in SEQID NO:22, SEQ ID NO:23, and/or SEQ ID NO:24.

In some embodiments, the (C₃₋₈ alkyl)-C(═O)OCH₃ ester can beenzymatically converted to the (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester usinga polypeptide having monooxygenase activity. In some embodiments, themonooxygenase is classified under EC 1.14.14.- or EC 1.14.15.-. In someembodiments, the monooxygenase can have at least 70% sequence identityto an amino acid sequence set forth in SEQ ID NO: 13-15, SEQ ID NO:27and/or SEQ ID NO:28.

In some embodiments, the C₄₋₉ carboxylic acid can be enzymaticallyproduced from a C₄₋₉ alkanoyl-CoA. In some embodiments, a polypeptidehaving thioesterase activity can enzymatically produce the C₄₋₉carboxylic acid from the C₄₋₉ alkanoyl-CoA. In some embodiments, thethioesterase can have at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:1, and/or SEQ ID NO: 32-33. In someembodiments, a polypeptide having butanal dehydrogenase activity and apolypeptide having aldehyde dehydrogenase activity enzymatically producethe C₄₋₉ carboxylic acid from C₄₋₉ alkanoyl-CoA.

This document also features methods of producing one or morehydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters. The methodincludes enzymatically converting a C₄₋₉ alkanoyl-CoA to a (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester; and enzymatically converting the (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester.

In some embodiments, the C₄₋₉ alkanoyl-CoA can be enzymaticallyconverted to the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having alcohol O-acetyltransferase activity. In someembodiments, the alcohol O-acetyltransferase can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO: 25.

In some embodiments, (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester isenzymatically converted to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having monooxygenase activity. In some embodiments, saidpolypeptide having monooxygenase activity is classified under EC1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further can include enzymaticallyconverting the (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester to a C₄₋₉hydroxyalkanoate. In some embodiments, a polypeptide having esteraseactivity enzymatically converts the (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester or (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester tothe C₄₋₉ hydroxyalkanoate.

In one aspect, this document features a method for producing abioderived 6-carbon compound. The method for producing a bioderived sixcarbon compound can include culturing or growing a recombinant host asdescribed herein under conditions and for a sufficient period of time toproduce the bioderived six carbon compound, wherein, optionally, thebioderived six carbon compound is selected from the group consisting ofadipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine,1,6-hexanediol, and combinations thereof.

In one aspect, this document features composition comprising abioderived six carbon compound as described herein and a compound otherthan the bioderived six carbon compound, wherein the bioderived sixcarbon compound is selected from the group consisting of adipic acid,adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoic acid,caprolactam, hexamethylenediamine, 1,6-hexanediol, and combinationsthereof. For example, the bioderived six carbon compound is a cellularportion of a host cell or an organism.

This document also features a biobased polymer comprising the bioderivedadipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine,1,6-hexanediol, and combinations thereof.

This document also features a biobased resin comprising the bioderivedadipic acid, adipate semialdehyde, 6-aminohexanoic acid,6-hydroxyhexanoic acid, caprolactam, hexamethylenediamine,1,6-hexanediol, and combinations thereof, as well as a molded productobtained by molding a biobased resin.

In another aspect, this document features a process for producing abiobased polymer that includes chemically reacting the bioderived adipicacid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoicacid, caprolactam, hexamethylenediamine, and/or 1,6-hexanediol, withitself or another compound in a polymer producing reaction.

In another aspect, this document features a process for producing abiobased resin that includes chemically reacting the bioderived adipicacid, adipate semialdehyde, 6-aminohexanoic acid, 6-hydroxyhexanoicacid, caprolactam, hexamethylenediamine, and/or 1,6-hexanediol, withitself or another compound in a resin producing reaction.

In another aspect, this document provides a bio-derived product,bio-based product or fermentation-derived product, wherein said productcomprises:

(i) a composition comprising at least one bio-derived, bio-based orfermentation-derived compound provided herein or in any one of FIGS.1-8, or any combination thereof;

(ii) a bio-derived, bio-based or fermentation-derived polymer comprisingthe bio-derived, bio-based or fermentation-derived composition orcompound of (i), or any combination thereof;

(iii) a bio-derived, bio-based or fermentation-derived resin comprisingthe bio-derived, bio-based or fermentation-derived compound orbio-derived, bio-based or fermentation-derived composition of (i) or anycombination thereof or the bio-derived, bio-based orfermentation-derived polymer of (ii) or any combination thereof;

(iv) a molded substance obtained by molding the bio-derived, bio-basedor fermentation-derived polymer of (ii) or the bio-derived, bio-based orfermentation-derived resin of (iii), or any combination thereof;

(v) a bio-derived, bio-based or fermentation-derived formulationcomprising the bio-derived, bio-based or fermentation-derivedcomposition of (i), bio-derived, bio-based or fermentation-derivedcompound of (i), bio-derived, bio-based or fermentation-derived polymerof (ii), bio-derived, bio-based or fermentation-derived resin of (iii),or bio-derived, bio-based or fermentation-derived molded substance of(iv), or any combination thereof; or

(vi) a bio-derived, bio-based or fermentation-derived semi-solid or anon-semi-solid stream, comprising the bio-derived, bio-based orfermentation-derived composition of (i), bio-derived, bio-based orfermentation-derived compound of (i), bio-derived, bio-based orfermentation-derived polymer of (ii), bio-derived, bio-based orfermentation-derived resin of (iii), bio-derived, bio-based orfermentation-derived formulation of (v), or bio-derived, bio-based orfermentation-derived molded substance of (iv), or any combinationthereof.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having (i) fatty acidO-methyltransferase activity or alcohol O-acetyltransferase activity,(ii) monooxygenase activity, and (iii) esterase or demethylase activity.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having fatty acidO-methyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the biochemical network enzymatically produces6-hydroxyhexanoate methyl ester. The biochemical network can furtherinclude a polypeptide having demethylase activity or a polypeptidehaving esterase activity, wherein the polypeptide having demethylaseactivity or a polypeptide having esterase activity enzymatically produce6-hydroxyhexanoate.

The biochemical network can further include at least one exogenousnucleic acid encoding a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity, wherein the polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity enzymatically produce C6 precursor molecules such ashexanoyl-CoA.

The biochemical network can further one or more of an exogenouspolypeptide having thioesterase activity, a polypeptide having aldehydedehydrogenase activity, or a polypeptide having butanal dehydrogenaseactivity, wherein the polypeptide having thioesterase activity, apolypeptide having aldehyde dehydrogenase activity, or a polypeptidehaving butanal dehydrogenase activity enzymatically produce hexanoate asa C6 precursor molecule.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having alcoholO-acetyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the biochemical network produces hexanoic acid hexylester. The biochemical network can further include an esterase, whereinthe esterase enzymatically converts hexanoic acid hexyl ester to6-hydroxyhexanoate and/or 1,6-hexanediol.

The biochemical network can further include at least one exogenousnucleic acid encoding a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity, wherein the polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity enzymatically produce C6 precursor molecules such ashexanoyl-CoA. The biochemical network can further include one or more ofan exogenous a polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activity,wherein the polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activityenzymatically produce hexanol as a C6 precursor molecule.

A biochemical network producing 6-hydroxyhexanoate can further includeone or more of a polypeptide having monooxygenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingaldehyde dehydrogenase activity, a polypeptide having 7-oxohexanoatedehydrogenase activity, a polypeptide having 6-oxohexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity or a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, wherein the polypeptide having monooxygenaseactivity, a polypeptide having alcohol dehydrogenase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity enzymatically convert6-hydroxyhexanoate to adipic acid or adipate semialdehyde.

A biochemical network producing 6-hydroxyhexanoate can further includeone or more of a polypeptide having ω-transaminase activity, apolypeptide having 6-hydroxyhexanoate dehydrogenase activity, apolypeptide having 5-hydroxybutanoate dehydrogenase activity, apolypeptide having 4-hydroxybutyrate dehydrogenase activity and apolypeptide having alcohol dehydrogenase activity, wherein thepolypeptide having ω-transaminase activity, a polypeptide having6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxybutanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity and a polypeptide havingalcohol dehydrogenase activity enzymatically convert 6-hydroxyhexanoateto 6-aminohexanoate.

A biochemical network producing 6-aminohexanoate, 6-hydroxyhexanoate,adipate semialdehyde, or 1,6-hexanediol can further include one or moreof a polypeptide having carboxylate reductase activity, a polypeptidehaving ω-transaminase activity, a polypeptide having deacetylaseactivity, a polypeptide having N-acetyl transferase activity, or apolypeptide having alcohol dehydrogenase activity, wherein the apolypeptide having carboxylate reductase activity, a polypeptide havingω-transaminase activity, a polypeptide having deacetylase activity, apolypeptide having N-acetyl transferase activity, or a polypeptidehaving alcohol dehydrogenase activity, enzymatically convert6-aminohexanoate, 6-hydroxyhexanoate, adipate semialdehyde, or1,6-hexanediol to hexamethylenediamine.

A biochemical network producing 6-hydroxyhexanoate can further includeone or more of a polypeptide having carboxylate reductase activity and apolypeptide having alcohol dehydrogenase activity, wherein thepolypeptide having carboxylate reductase activity and a polypeptidehaving alcohol dehydrogenase activity enzymatically convert6-hydroxyhexanoic acid to 1,6-hexanediol.

Also, described herein is a means for obtaining 6-hydroxyhexanoate using(i) a polypeptide having fatty acid O-methyltransferase activity and apolypeptide having monooxygenase activity and (ii) a polypeptide havingdemethylase activity or a polypeptide having esterase activity. Themeans can further include means for converting 6-hydroxyhexanoate to atleast one of adipic acid, 6-aminohexanoic acid, caprolactam,hexamethylenediamine, 6-hydroxyhexanoic acid, and 1,6-hexanediol. Themeans can include a polypeptide having aldehyde dehydrogenase activity,a polypeptide having 7-oxohexanoate dehydrogenase activity, apolypeptide having 6-oxohexanoate dehydrogenase activity, a polypeptidehaving 5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity.

Also, described herein is a means for obtaining 6-hydroxyhexanoate using(i) a polypeptide having alcohol O-acetyltransferase and a polypeptidehaving monooxygenase activity and (ii) a polypeptide having demethylaseactivity or a polypeptide having esterase activity. The means canfurther include means for converting 6-hydroxyhexanoate to at least oneof adipic acid, 6-aminohexanoic acid, caprolactam, hexamethylenediamine,6-hydroxyhexanoic acid, and 1,6-hexanediol. The means can include apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity.

Also described herein is (i) a step for obtaining 6-hydroxyhexanoateusing a polypeptide having alcohol O-acetyltransferase, a polypeptidehaving monooxygenase activity, and a polypeptide having demethylaseactivity or a polypeptide having esterase activity, and (ii) a step forobtaining adipic acid, 6-aminohexanoate, adipate semialdehyde1,6-hexanediol, hexamethylenediamine using a polypeptide havingcarboxylate reductase activity, a polypeptide having alcoholdehydrogenase activity, a polypeptide having ω-transaminase activity, apolypeptide having deacetylase activity, a polypeptide having N-acetyltransferase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxybutanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, a polypeptide having aldehyde dehydrogenaseactivity, a polypeptide having 7-oxohexanoate dehydrogenase activity, apolypeptide having 6-oxohexanoate dehydrogenase activity, a polypeptidehaving 5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity,

In another aspect, this document features a composition comprising6-hydroxyhexanoate and a polypeptide having alcohol O-acetyltransferase,a polypeptide having monooxygenase activity, and a polypeptide havingdemethylase activity or a polypeptide having esterase activity complex.The composition can be cellular. The composition can further include apolypeptide having carboxylate reductase activity, a polypeptide havingalcohol dehydrogenase activity, a polypeptide having ω-transaminaseactivity, a polypeptide having deacetylase activity, a polypeptidehaving N-acetyl transferase activity, a polypeptide having6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxybutanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity, a polypeptide having aldehydedehydrogenase activity, a polypeptide having 7-oxohexanoatedehydrogenase activity, a polypeptide having 6-oxohexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity or a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, and at least one of adipic acid, 6-aminohexanoicacid, caprolactam, hexamethylenediamine, 6-hydroxyhexanoic acid, and1,6-hexanediol. The composition can be cellular.

The reactions of the pathways described herein can be performed in oneor more cell (e.g., host cell) strains (a) naturally expressing one ormore relevant enzymes, (b) genetically engineered to express one or morerelevant enzymes, or (c) naturally expressing one or more relevantenzymes and genetically engineered to express one or more relevantenzymes. Alternatively, relevant enzymes can be extracted from of theabove types of host cells and used in a purified or semi-purified form.Extracted enzymes can optionally be immobilized to the floors and/orwalls of appropriate reaction vessels. Moreover, such extracts includelysates (e.g. cell lysates) that can be used as sources of relevantenzymes. In the methods provided by the document, all the steps can beperformed in cells (e.g., host cells), all the steps can be performedusing extracted enzymes, or some of the steps can be performed in cellsand others can be performed using extracted enzymes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein including GenBank and NCBI submissions with accessionnumbers are incorporated by reference in their entirety. In case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and the drawings, and from the claims. The word “comprising”in the claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of exemplary biochemical pathways leading tohexanoyl-CoA using NADH-dependent enzymes and acetyl-CoA as centralmetabolites.

FIG. 2 is a schematic of exemplary biochemical pathways leading tohexanoyl-CoA using NADPH-dependent enzymes and acetyl-CoA as centralmetabolites.

FIG. 3 is a schematic of exemplary biochemical pathways leading tohexanoate and hexanol using hexanoyl-CoA as a central precursor.

FIG. 4 is a schematic of an exemplary biochemical pathway leading toadipic acid using 6-hydroxyhexanoate as a central precursor.

FIG. 5 is a schematic of exemplary biochemical pathways leading to6-aminohexanoate and caprolactam using 6-hydroxyhexanoate as a centralprecursor.

FIG. 6 is a schematic of exemplary biochemical pathways leading tohexamethylenediamine using 6-aminohexanoate, 6-hydroxyhexanoate, adipatesemialdehyde or 1,6-hexanediol as a central precursor.

FIG. 7 is a schematic of exemplary biochemical pathways leading to6-hydroxyhexanoate via ester intermediates using hexanoate orhexanoyl-CoA as a central precursor. FIG. 7 also contains an exemplarybiochemical pathway leading to 6-hydroxyhexanoate using 1,6-hexanediolas a central precursor.

FIG. 8 is a schematic of exemplary biochemical pathways leading to1,6-hexanediol using 6-hydroxyhexanoate or hexanoyl-CoA as a centralprecursor.

FIG. 9 contains the amino acid sequences of an Escherichia colithioesterase encoded by tesB (See GenBank Accession No. AAA24665.1, SEQID NO: 1), a Mycobacterium marinum carboxylate reductase (See GenbankAccession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatiscarboxylate reductase (See Genbank Accession No. ABK71854.1, SEQ ID NO:3), a Segniliparus rugosus carboxylate reductase (See Genbank AccessionNo. EFV11917.1, SEQ ID NO: 4), a Mycobacterium massiliense carboxylatereductase (See Genbank Accession No. EIV11143.1, SEQ ID NO: 5), aSegniliparus rotundus carboxylate reductase (See Genbank Accession No.ADG98140.1, SEQ ID NO: 6), a Chromobacterium violaceum ω-transaminase(See Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa ω-transaminase (See Genbank Accession No. AAG08191.1, SEQ IDNO: 8), a Pseudomonas syringae ω-transaminase (See Genbank Accession No.AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides ω-transaminase (SeeGenbank Accession No. ABA81135.1, SEQ ID NO: 10), an Escherichia coliw-transaminase (See Genbank Accession No. AAA57874.1, SEQ ID NO: 11) anda Vibrio Fluvialis ω-transaminase (See Genbank Accession No. AEA39183.1,SEQ ID NO: 12), a Polaromonas sp. JS666 monooxygenase (See GenbankAccession No. ABE47160.1, SEQ ID NO:13), a Mycobacterium sp. HXN-1500monooxygenase (See Genbank Accession No. CAH04396.1, SEQ ID NO:14), aMycobacterium austroafricanum monooxygenase (See Genbank Accession No.ACJ06772.1, SEQ ID NO:15), a Polaromonas sp. JS666 oxidoreductase (SeeGenbank Accession No. ABE47159.1, SEQ ID NO:16), a Mycobacterium sp.HXN-1500 oxidoreductase (See Genbank Accession No. CAH04397.1, SEQ IDNO:17), a Polaromonas sp. JS666 ferredoxin (See Genbank Accession No.ABE47158.1, SEQ ID NO:18), a Mycobacterium sp. HXN-1500 ferredoxin (SeeGenbank Accession No. CAH04398.1, SEQ ID NO:19), Bacillus subtilisphosphopantetheinyl transferase (See Genbank Accession No. CAA44858.1,SEQ ID NO:20), Nocardia sp. NRRL 5646 phosphopantetheinyl transferase(See Genbank Accession No. ABI83656.1, SEQ ID NO:21), a Mycobacteriummarinum fatty acid O-methyltransferase (GenBank Accession No.ACC41782.1; SEQ ID NO:22), a Mycobacterium smegmatis str. MC2 fatty acidO-methyltransferase (GenBank Accession No. ABK73223.1; SEQ ID NO:23), aPseudomonas putida fatty acid O-methyltransferase (GenBank Accession No.CAA39234.1; SEQ ID NO:24), a Saccharomyces cerevisiae alcoholO-acetyltransferase (Genbank Accession No: CAA85138.1, SEQ ID NO: 25), aPseudomonas fluorescens carboxylesterase (Genbank Accession No.AAC60471.2, SEQ ID NO: 26), a Pseudomonas putida alkane 1-monooxygenase(Genbank Accession No. CAB51047.1, SEQ ID NO: 27), a Candida maltosecytochrome P450 (Genbank Accession Nos: BAA00371.1, SEQ ID NO: 28), aSalmonella enterica subsp. enterica serovar Typhimurium butanaldehydrogenase (GenBank Accession No. AAD39015, SEQ ID NO:29), aSphingomonas paucimobilis demethylase (GenBank Accession No. BAD61059.1and GenBank Accession No. BAC79257.1, SEQ ID NOs: 30 and 31,respectively), a Lactobacillus brevis thioesterase (GenBank AccessionNo. ABJ63754.1, SEQ ID NO:32), and a Lactobacillus plantarumthioesterase (GenBank Accession No. CCC78182.1, SEQ ID NO:33).

FIG. 10 is a bar graph of the relative absorbance at 412 nm of releasedCoA as a measure of the activity of a thioesterase for convertinghexanoyl-CoA to hexanoate relative to the empty vector control.

FIG. 11 is a bar graph summarizing the change in absorbance at 340 nmafter 20 min, which is a measure of the consumption of NADPH and theactivity of the carboxylate reductases with no substrate (enzyme onlycontrols).

FIG. 12 is a bar graph of the change in absorbance at 340 nm after 20min, which is a measure of the consumption of NADPH and the activity ofcarboxylate reductases for converting adipate to adipate semialdehyderelative to the empty vector control.

FIG. 13 is a bar graph of the change in absorbance at 340 nm after 20min, which is a measure of the consumption of NADPH and the activity ofcarboxylate reductases for converting 6-hydroxyhexanoate to6-hydroxyhexanal relative to the empty vector control.

FIG. 14 is a bar graph of the change in absorbance at 340 nm after 20min, which is a measure of the consumption of NADPH and the activity ofcarboxylate reductases for converting N6-acetyl-6-aminohexanoate toN6-acetyl-6-aminohexanal relative to the empty vector control.

FIG. 15 is a bar graph of the change in absorbance at 340 nm after 20min, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases for converting adipate semialdehyde to hexanedialrelative to the empty vector control.

FIG. 16 is a bar graph summarizing the percent conversion after 4 hoursof pyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of the enzyme only controls (no substrate).

FIG. 17 is a bar graph of the percent conversion after 24 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 6-aminohexanoate to adipate semialdehyderelative to the empty vector control.

FIG. 18 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity for converting adipate semialdehyde to 6-aminohexanoaterelative to the empty vector control.

FIG. 19 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting hexamethylenediamine to 6-aminohexanal relativeto the empty vector control.

FIG. 20 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting N6-acetyl-1,6-diaminohexane toN6-acetyl-6-aminohexanal relative to the empty vector control.

FIG. 21 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 6-aminohexanol to 6-oxohexanol relative to theempty vector control.

FIG. 22 is a bar graph of the change in peak area for 6-hydroxyhexanoateas determined via LC-MS, as a measure of the monooxygenase activity forconverting hexanoate to 6-hydroxyhexanoate relative to the empty vectorcontrol.

FIG. 23 is a bar graph of the change in absorbance at 340 nm after 20min, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases for converting hexanoic acid to hexanal relativeto the empty vector control.

DETAILED DESCRIPTION

This document provides enzymes, non-natural pathways, cultivationstrategies, feedstocks, central precursors, host microorganisms andattenuations to the host's biochemical network, which generate a sixcarbon chain aliphatic backbone (which can be bound to a coenzyme Amoiety) from central metabolites in which two terminal functional groupsmay be formed leading to the synthesis of one or more of adipic acid,6-hydroxyhexanoate, 6-aminohexanoate, caprolactam, hexamethylenediamineor 1,6-hexanediol (referred to as “C6 building blocks” herein). As usedherein, the term “central precursor” is used to denote any metabolite inany metabolic pathway shown herein leading to the synthesis of a C6building block. The term “central metabolite” is used herein to denote ametabolite that is produced in all microorganisms to support growth.

Host microorganisms described herein can include endogenous pathwaysthat can be manipulated such that one or more C6 building blocks orcentral precursors thereof can be produced. In an endogenous pathway,the host microorganism naturally expresses all of the enzymes catalyzingthe reactions within the pathway. A host microorganism containing anengineered pathway does not naturally express all of the enzymescatalyzing the reactions within the pathway but has been engineered suchthat all of the enzymes within the pathway are expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once thatchromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or other compoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

For example, depending on the host and the compounds produced by thehost, one or more of the following polypeptides may be expressed in thehost in addition to a polypeptide having fatty acid O-methyltransferaseactivity or a polypeptide having alcohol O-acetyltransferase activity: apolypeptide having monooxygenase activity, a polypeptide having esteraseactivity, a polypeptide having demethylase activity, a polypeptidehaving β-ketothiolase activity, a polypeptide having acetyl-CoAcarboxylase activity, a polypeptide having β-ketoacyl-[acp] synthaseactivity, a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity,a polypeptide having 3-oxoacyl-CoA reductase activity, a polypeptidehaving enoyl-CoA hydratase activity, a polypeptide havingtrans-2-enoyl-CoA reductase activity, a polypeptide having thioesteraseactivity, a polypeptide having aldehyde dehydrogenase activity, apolypeptide having butanal dehydrogenase activity, a polypeptide havingmonooxygenase activity in, for example, the CYP4F3B family, apolypeptide having alcohol dehydrogenase activity, a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide havingω-transaminase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxybutanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, a polypeptide having carboxylate reductaseactivity, a polypeptide having deacetylase activity, a polypeptidehaving N-acetyl transferase activity, or a polypeptide havingamidohydrolase activity. In recombinant hosts expressing a polypeptidehaving carboxylate reductase activity, a polypeptide havingphosphopantetheinyl transferase activity also can be expressed as itenhances activity of the polypeptide having carboxylate reductaseactivity. In recombinant hosts expressing a polypeptide havingmonooxygenase activity, an electron transfer chain protein such as apolypeptide having oxidoreductase activity and/or a polypeptide havingferredoxin polypeptide activity also can be expressed.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having (i) fatty acidO-methyltransferase activity or a polypeptide having alcoholO-acetyltransferase activity, (ii) a polypeptide having monooxygenaseactivity, and (iii) a polypeptide having esterase activity or apolypeptide having demethylase activity.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having fatty acidO-methyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the host produces 6-hydroxyhexanoate methyl ester.Such a host further can include a polypeptide having demethylaseactivity or a polypeptide having esterase activity and further produce6-hydroxyhexanoate. In some embodiments, the recombinant host also caninclude at least one exogenous nucleic acid encoding a polypeptidehaving β-ketothiolase activity or a polypeptide having acetyl-CoAcarboxylase activity and a polypeptide having β-ketoacyl-[acp] synthaseactivity, a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activityor a polypeptide having 3-oxoacyl-CoA reductase activity, a polypeptidehaving enoyl-CoA hydratase activity, and a polypeptide havingtrans-2-enoyl-CoA reductase activity to produce C6 precursor moleculessuch as hexanoyl-CoA. Such a host further can include one or more of(e.g., two or three of) an exogenous polypeptide having thioesteraseactivity, a polypeptide having aldehyde dehydrogenase activity, or apolypeptide having butanal dehydrogenase activity, and produce hexanoateas a C6 precursor molecule.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having alcoholO-acetyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the host produces hexanoic acid hexyl ester. Such ahost further can include a polypeptide having esterase activity andfurther produce 6-hydroxyhexanoate and/or 1,6-hexanediol. In someembodiments, the recombinant host also can include at least oneexogenous nucleic acid encoding a polypeptide having β-ketothiolaseactivity or a polypeptide having acetyl-CoA carboxylase activity and apolypeptide having β-ketoacyl-[acp] synthase activity, a polypeptidehaving 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity to produce C6 precursor molecules such as hexanoyl-CoA. Such ahost further can include one or more of (e.g., two or three of) anexogenous a polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activity andproduce hexanol as a C6 precursor molecule.

A recombinant host producing 6-hydroxyhexanoate further can include oneor more of a polypeptide having monooxygenase activity (e.g., in theCYP4F3B family), a polypeptide having alcohol dehydrogenase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity, and produce adipic acid oradipate semialdehyde. For example, a recombinant host further caninclude a polypeptide having monooxygenase activity and produce adipicacid or adipate semialdehyde. As another example, a recombinant hostfurther can include (i) a polypeptide having alcohol dehydrogenaseactivity, a polypeptide having 6-hydroxyhexanoate dehydrogenaseactivity, a polypeptide having 5-hydroxybutanoate dehydrogenaseactivity, or a polypeptide having 4-hydroxybutyrate dehydrogenaseactivity or (ii) a polypeptide having aldehyde dehydrogenase activity, apolypeptide having 5-oxobutanoate dehydrogenase activity, a polypeptidehaving 6-oxohexanoate dehydrogenase activity, or a polypeptide having7-oxohexanoate dehydrogenase activity, and produce adipic acid.

A recombinant host producing 6-hydroxyhexanoate further can include oneor more of a polypeptide having transaminase activity, a polypeptidehaving 6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxybutanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity and a polypeptide havingalcohol dehydrogenase activity, and produce 6-aminohexanoate. Forexample, a recombinant host producing 6-hydroxyhexanoate further caninclude a polypeptide having ω-transaminase activity and either apolypeptide having 6-hydroxyhexanoate dehydrogenase activity orpolypeptide having alcohol dehydrogenase activity.

A recombinant host producing 6-aminohexanoate, 6-hydroxyhexanoate,adipate semialdehyde or 1,6-hexanediol further can include one or moreof a polypeptide having carboxylate reductase activity, a polypeptidehaving ω-transaminase activity, a polypeptide having deacetylaseactivity, a polypeptide having N-acetyl transferase activity, or apolypeptide having alcohol dehydrogenase activity, and producehexamethylenediamine. In some embodiments, a recombinant host furthercan include each of a polypeptide having carboxylate reductase activity,a polypeptide having ω-transaminase activity, a polypeptide havingdeacetylase activity, and a polypeptide having N-acetyl transferaseactivity. In some embodiments, a recombinant host further can include apolypeptide having carboxylate reductase activity and a polypeptidehaving ω-transaminase activity. In some embodiments, a recombinant hostfurther can include a polypeptide having carboxylate reductase activity,a polypeptide having ω-transaminase activity, and a polypeptide havingalcohol dehydrogenase activity. In the embodiments in which therecombinant host produces 6-aminohexanoate, an additional polypeptidehaving ω-transaminase activity may not be necessary to producehexamethylenediamine. In some embodiments, the host includes a secondexogenous polypeptide having ω-transaminase activity that differs fromthe first exogenous polypeptide having ω-transaminase activity.

A recombinant host producing 6-hydroxyhexanoic acid further can includeone or more of a polypeptide having carboxylate reductase activity and apolypeptide having alcohol dehydrogenase activity, and produce1,6-hexanediol.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species or genus, or can be from multiple sources, i.e.,different species or genera. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank orEMBL. In recombinant hosts containing an exogenous enzyme, the hostscontain an exogenous nucleic acid encoding the enzyme.

Any of the enzymes described herein that can be used for production ofone or more C6 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of thecorresponding wild-type enzyme. It will be appreciated that the sequenceidentity can be determined on the basis of the mature enzyme (e.g., withany signal sequence removed) or on the basis of the immature enzyme(e.g., with any signal sequence included).

For example, a polypeptide having thioesterase activity described hereincan have at least 70% sequence identity (homology) (e.g., at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) tothe amino acid sequence of an Escherichia coli thioesterase encoded bytesB (see GenBank Accession No. AAA24665.1, SEQ ID NO: 1), to the aminoacid sequence of a Lactobacillus brevis thioesterase (GenBank AccessionNo. ABJ63754.1, SEQ ID NO:32) or a Lactobacillus plantarum thioesterase(GenBank Accession No. CCC78182.1, SEQ ID NO:33). See FIG. 9.

For example, a polypeptide having carboxylate reductase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marinum(see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacteriumsmegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), aSegniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO:4), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1,SEQ ID NO: 5), or a Segniliparus rotundus (see Genbank Accession No.ADG98140.1, SEQ ID NO: 6) carboxylate reductase. See, FIG. 9.

For example, a polypeptide having ω-transaminase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQID NO: 10), an Escherichia coli (see Genbank Accession No. AAA57874.1,SEQ ID NO: 11), or a Vibrio fluvialis (see Genbank Accession No.AEA39183.1, SEQ ID NO: 12) ω-transaminase. Some of these ω-transaminasesare diamine ω-transaminases. See, FIG. 9.

For example, a polypeptide having monooxygenase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Polaromonas sp. JS666monooxygenase (see Genbank Accession No. ABE47160.1, SEQ ID NO:13), aMycobacterium sp. HXN-1500 monooxygenase (see Genbank Accession No.CAH04396.1, SEQ ID NO:14), or a Mycobacterium austroafricanummonooxygenase (See Genbank Accession No. ACJ06772.1, SEQ ID NO:15). See,FIG. 9.

For example, a polypeptide having oxidoreductase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Polaromonas sp. JS666oxidoreductase (see Genbank Accession No. ABE47159.1, SEQ ID NO:16) or aMycobacterium sp. HXN-1500 oxidoreductase (see Genbank Accession No.CAH04397.1, SEQ ID NO:17). See, FIG. 9.

For example, a polypeptide having ferredoxin activity described hereincan have at least 70% sequence identity (homology) (e.g., at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) tothe amino acid sequence of a Polaromonas sp. JS666 ferredoxin (seeGenbank Accession No. ABE47158.1, SEQ ID NO:18) or a Mycobacterium sp.HXN-1500 ferredoxin (see Genbank Accession No. CAH04398.1, SEQ IDNO:19). See, FIG. 9.

For example, a polypeptide having phosphopantetheinyl transferaseactivity described herein can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillussubtilis phosphopantetheinyl transferase (see Genbank Accession No.CAA44858.1, SEQ ID NO:20) or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:21). See FIG. 9.

For example, a polypeptide having fatty acid O-methyltransferaseactivity described herein can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aMycobacterium marinum (see GenBank Accession No. ACC41782.1, SEQ ID NO:22), a Mycobacterium smegmatis (see GenBank Accession No. ABK73223.1,SEQ ID NO: 23), or a Pseudomonas putida (see GenBank Accession No.CAA39234.1, SEQ ID NO: 24) methyltransferase. See FIG. 9.

For example, a polypeptide having alcohol O-acetyltransferase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Saccharomycescerevisiae (see GenBank Accession No. CAA85138.1, SEQ ID NO: 25) alcoholO-acetyltransferase. See FIG. 9.

For example, a polypeptide having esterase activity described herein canhave at least 70% sequence identity (homology) (e.g., at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to theamino acid sequence of a Pseudomonas fluorescens (see GenBank AccessionNo. AAC60471.2, SEQ ID NO: 26) esterase. See FIG. 9.

For example, a polypeptide having alkane 1-monooxygenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Pseudomonas putidaalkane 1-monooxygenase (Genbank Accession No. CAB51047.1, SEQ ID NO:27).

For example, a polypeptide having cytochrome P450 monooxygenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Candida maltosecytochrome P450 (Genbank Accession No: BAA00371.1, SEQ ID NO: 28).

For example, a polypeptide having butanal dehydrogenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Salmonella entericasubsp. enterica serovar Typhimurium butanal dehydrogenase (see GenBankAccession No. AAD39015, SEQ ID NO:29).

For example, a polypeptide having syringate O-demethylase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Sphingomonaspaucimobilis demethylase (see, GenBank Accession No. BAD61059.1 andGenBank Accession No. BAC79257.1, SEQ ID NOs: 30 and 31, respectively).

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\B12seq-i c:\seq1.txt-jc:\seq2.txt-p blastp-o c:\output.txt. If the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that is shorterthan the full-length immature protein and has at least 25% (e.g., atleast: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 91%; 92%; 93%; 94%;95%; 96%; 97%; 98%; 99%; 100%; or even greater than 100%) of theactivity of the corresponding mature, full-length, wild-type protein.The functional fragment can generally, but not always, be comprised of acontinuous region of the protein, wherein the region has functionalactivity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered hosts can naturally express none or some (e.g., one or more,two or more, three or more, four or more, five or more, or six or more)of the enzymes of the pathways described herein. Thus, a pathway withinan engineered host can include all exogenous enzymes, or can includeboth endogenous and exogenous enzymes. Endogenous genes of theengineered hosts also can be disrupted to prevent the formation ofundesirable metabolites or prevent the loss of intermediates in thepathway through other enzymes acting on such intermediates. Engineeredhosts can be referred to as recombinant hosts or recombinant host cells.As described herein recombinant hosts can include nucleic acids encodingone or more of a polypeptide having fatty acid O-methyltransferaseactivity, a polypeptide having alcohol O-acetyltransferase activity, apolypeptide having dehydrogenase activity, a polypeptide havingβ-ketothiolase activity, a polypeptide having β-ketoacyl-[acp] synthaseactivity, a polypeptide having carboxylase activity, a polypeptidehaving reductase activity, a polypeptide having hydratase activity, apolypeptide having thioesterase activity, a polypeptide havingmonooxygenase activity, a polypeptide having demethylase activity, apolypeptide having esterase activity, or a polypeptide havingtransaminase activity as described herein.

In addition, the production of one or more C6 building blocks can beperformed in vitro using the isolated enzymes described herein, using alysate (e.g., a cell lysate) from a host microorganism as a source ofthe enzymes, or using a plurality of lysates from different hostmicroorganisms as the source of the enzymes.

Biosynthetic Methods

The present document provides methods of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester. As used herein, the term (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester refers to a compound having the followingformula:

As used herein, the term “C₃₋₈ hydroxyalkyl” refers to a saturatedhydrocarbon group that may be straight-chain or branched, and issubstituted by at least one hydroxyl (i.e., hydroxy or OH) group. Insome embodiments, the C₃₋₈ hydroxyalkyl refers to refers to a saturatedhydrocarbon group that may be straight-chain or branched, and issubstituted by at least one terminal hydroxyl (OH) group. In someembodiments, the alkyl group contains 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8 carbon atoms. In someembodiments, the C₃₋₈ hydroxyalkyl is a group of the following formula:

In some embodiments, the method comprises:

a) enzymatically converting a C₃₋₈ carboxylic acid to a (C₃₋₈alkyl)-C(═O)OCH₃ ester; and

b) enzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

As used herein, the term “C₄₋₉ carboxylic acid” refers to a compoundhaving the formula R—C(═O)OH, wherein R is a refers to a saturatedhydrocarbon group (i.e., an alkyl group) that may be straight-chain orbranched, wherein the compound has from 4 to 9 carbon atoms. In someembodiments, the C₄₋₉ carboxylic acid group contains 4 to 9, 4 to 8, 4to 7, 4 to 6, 4 to 5, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 9, 6 to 8, 6to 7, 7 to 9, 7 to 8, or 8 to 9 carbon atoms. Exemplary C₄₋₉ carboxylicacids include butanoic acid (i.e., butanoate), pentanoic acid (i.e.,pentanoate), hexanoic acid (i.e., hexanoate), heptanoic acid (i.e.,heptanoate), octanoic acid (e.g., octanoate), nonanoic acid (i.e.,nonanoate), 2-methylhexanoic acid (i.e., 2-methylhexanoate),3-methylhexanoic acid (i.e., 3-methylhexanoate), 4-methylhexanoic acid(i.e., 4-methylhexanoate), and 5-methylhexanoic acid (i.e.,5-methylhexanoate). In some embodiments, the C₄₋₉ carboxylic acid ishexanoate (i.e., hexanoic acid).

As used herein, the term “(C₃₋₈ alkyl)-C(═O)OCH₃ ester” refers to acompound having the following formula:

As used herein, the term “C₃₋₈ alkyl” refers to a saturated hydrocarbongroup that may be straight-chain or branched, having 3 to 9 carbonatoms. In some embodiments, the alkyl group contains 4 to 8, 4 to 7, 4to 6, 4 to 5, 5 to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8, carbonatoms. Example alkyl moieties include n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, iso-pentyl, neo-pentyl, n-hexyl, n-heptyl,n-octyl. In some embodiments, the C₃₋₈ alkyl is a group of the followingformula:

In some embodiments, the method comprises:

a) enzymatically converting a C₄₋₉ carboxylic acid to a (C₃₋₈alkyl)-C(═O)OCH₃ ester; and

b) enzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

In some embodiments, the C₄₋₉ carboxylic acid is enzymatically convertedto the (C₃₋₈ alkyl)-C(═O)OCH₃ ester using a polypeptide having fattyacid O-methyltransferase activity. In some embodiments, the polypeptidehaving fatty acid O-methyltransferase activity has at least 70% sequenceidentity to an amino acid sequence set forth in SEQ ID NO:23, SEQ IDNO:24, or SEQ ID NO:25.

In some embodiments, the (C₃₋₈ alkyl)-C(═O)OCH₃ ester is enzymaticallyconverted to the (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester using a polypeptidehaving monooxygenase activity. In some embodiments, the monooxygenase isclassified under EC 1.14.14.- or EC 1.14.15.-. In some embodiments, themonooxygenase has at least 70% sequence identity to an amino acidsequence set forth in SEQ ID NO: 13-15, SEQ ID NO:27 and/or, SEQ IDNO:28.

In some embodiments, the C₄₋₉ carboxylic acid is enzymatically producedfrom C₄₋₉ alkanoyl-CoA. As used herein, the term “C₄₋₉ alkanoyl-CoA”refers to a compound having the following formula:

wherein the C₃₋₈ alkyl group is as defined herein. In some embodiments,the C₃₋₈ alkyl is a group of the following formula:

In some embodiments, a polypeptide having thioesterase activityenzymatically produces the C₄₋₉ carboxylic acid from C₄₋₉ alkanoyl-CoA.In some embodiments, the thioesterase has at least 70% sequence identityto the amino acid sequence set forth in SEQ ID NO:1.

In some embodiments, a polypeptide having butanal dehydrogenase activityand a polypeptide having aldehyde dehydrogenase activity enzymaticallyproduces the C₄₋₉ carboxylic acid from the C₄₋₉ alkanoyl-CoA.

In some embodiments, the method of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester is a method of producing6-hydroxyhexanoate methyl ester, said method comprising:

a) enzymatically converting hexanoate to hexanoate methyl ester; and

b) enzymatically converting the hexanoate methyl ester to6-hydroxyhexanoate methyl ester.

In some embodiments, hexanoate is enzymatically converted to hexanoatemethyl ester using a polypeptide having fatty acid O-methyltransferaseactivity. In some embodiments, the polypeptide having fatty acidO-methyltransferase activity has at least 70% sequence identity to anamino acid sequence set forth in SEQ ID NO:22, SEQ ID NO:23, or SEQ IDNO:24.

In some embodiments, hexanoate methyl ester is enzymatically convertedto 6-hydroxyhexanoate methyl ester using a polypeptide havingmonooxygenase activity. In some embodiments, the polypeptide havingmonooxygenase activity has at least 70% sequence identity to an aminoacid sequence set forth in SEQ ID NO: 13-15, SEQ ID NO:27 and/or, SEQ IDNO:28. In some embodiments, the polypeptide having monooxygenaseactivity is classified under EC 1.14.14.- or EC 1.14.15.-.

In some embodiments, hexanoate is enzymatically produced fromhexanoyl-CoA. In some embodiments, a polypeptide having thioesteraseactivity enzymatically produces hexanoate from hexanoyl-CoA. In someembodiments, the polypeptide having thioesterase activity has at least70% sequence identity to the amino acid sequence set forth in SEQ IDNO:1, and/or SEQ ID NO: 32-33.

In some embodiments, a polypeptide having butanal dehydrogenase activityand a polypeptide having aldehyde dehydrogenase activity enzymaticallyproduces hexanoate from hexanoyl-CoA.

The present document further provides methods of producing one or moreterminal hydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters. Asused herein, the term (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester” refers toa compound having the following formula:

wherein the C₃₋₈ alkyl group is as defined herein. As used herein theterm “terminal hydroxy-substituted “(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl)ester” refers to a compound having the following formula:

wherein at least one of the alkyl groups (i.e., at least one of the C₄₋₉alkyl and C₃₋₈ alkyl groups) is substituted by at least one terminalhydroxy (—OH) group, and the C₃₋₈ alkyl is as defined herein. As usedherein, the term “C₄₋₉ alkyl” refers to a saturated hydrocarbon groupthat may be straight-chain or branched, having 4 to 9 carbon atoms. Insome embodiments, the alkyl group contains 4 to 9, 4 to 8, 4 to 7, 4 to6, 4 to 5, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 9, 6 to 8, 6 to 7, 7 to9, 7 to 8, or 8 to 9 carbon atoms. Example alkyl moieties includen-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl,neo-pentyl, n-hexyl, n-heptyl, n-octyl, and n-nonyl. In someembodiments, one of the alkyl groups is substituted by at least oneterminal hydroxy group. In some embodiments, each of the alkyl groups issubstituted by at least one terminal hydroxy group. In some embodiments,one of the alkyl groups is substituted by one terminal hydroxy group. Insome embodiments, each of the alkyl groups is substituted by oneterminal hydroxy group. In some embodiments, the terminalhydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester is selectedfrom the group consisting of:

In some embodiments, the method includes:

a) enzymatically converting C₄₋₉ alkanoyl-CoA to a (C₄₋₉alkyl)-OC(═O)—(C3-8 alkyl) ester; and

b) enzymatically converting the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esterto any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, or (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester.

In some embodiments, C₄₋₉ alkanoyl-CoA is enzymatically converted to the(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester using a polypeptide havingalcohol O-acetyltransferase activity. In some embodiments, the alcoholO-acetyltransferase has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 25.

In some embodiments, the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester isenzymatically converted to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having monooxygenase activity. In some embodiments, thepolypeptide having monooxygenase activity is classified under EC1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further includes enzymaticallyconverting (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester to a C₄₋₉ hydroxyalkanoate. Insome embodiments, a polypeptide having esterase activity enzymaticallyconverts (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester to the C₄₋₉ hydroxyalkanoate.

As used herein, the term C₄₋₉ hydroxyalkanoate refers to a compoundhaving the following formula:

wherein the C₃₋₈ hydroxyalkyl is as defined herein. Example C₄₋₉hydroxyalkanoates include, but are not limited to, 6-hydroxyhexanoate(i.e., 6-hydroxyhexanoic acid), 5-hydroxyhexanoate (i.e.,5-hydroxyhexanoic acid), 4-hydroxyhexanoate (i.e., 4-hydroxyhexanoicacid), 3-hydroxyhexanoate (i.e., 3-hydroxyhexanoic acid), and the like.It is understood by those skilled in the art that the specific form willdepend on pH (e.g., neutral or ionized forms, including any salt formsthereof). In some embodiments, the C₃₋₈ hydroxyalkyl is a group havingthe following formula:

In some embodiments, the method of producing one or morehydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters is a methodof producing one or more hexanoic acid hexyl hydroxyl esters. In someembodiments, the method includes:

a) enzymatically converting hexanoyl-CoA to hexanoic acid hexyl ester;and

b) enzymatically converting hexanoic acid hexyl ester to any of6-hydroxyhexanoic acid hexyl ester, 6-hydroxyhexanoic acid6-hydroxyhexyl ester, or hexanoic acid 6-hydroxyhexyl ester.

In some embodiments, hexanoyl-CoA is enzymatically converted to hexanoicacid hexyl ester using a polypeptide having alcohol O-acetyltransferaseactivity. In some embodiments, the polypeptide having alcoholO-acetyltransferase activity has at least 70% sequence identity to theamino acid sequence set forth in SEQ ID NO: 25.

In some embodiments, hexanoic acid hexyl ester is enzymaticallyconverted to any of 6-hydroxyhexanoic acid hexyl ester,6-hydroxyhexanoic acid 6-hydroxyhexyl ester and/or hexanoic acid6-hydroxyhexyl ester using a polypeptide having monooxygenase activity.In some embodiments, the polypeptide having monooxygenase activity isclassified under EC 1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further includes enzymaticallyconverting 6-hydroxyhexanoic acid 6-hydroxyhexyl ester or6-hydroxyhexanoic acid hexyl ester to 6-hydroxyhexanoate. In someembodiments, a polypeptide having esterase activity enzymaticallyconverts 6-hydroxyhexanoic acid 6-hydroxyhexyl ester or6-hydroxyhexanoic acid hexyl ester to 6-hydroxyhexanoate.

In some embodiments, the method further includes enzymaticallyconverting 6-hydroxyhexanoic acid 6-hydroxyhexyl ester or hexanoic acid6-hydroxyhexyl ester to 1,6-hexanediol. In some embodiments, apolypeptide having esterase activity enzymatically converts6-hydroxyhexanoic acid 6-hydroxyhexyl ester or hexanoic acid6-hydroxyhexyl ester to 1,6-hexanediol.

In some embodiments, the method can include enzymatically converting6-hydroxyhexanoic acid hexyl ester, 6-hydroxyhexanoic acid6-hydroxyhexyl ester, or hexanoic acid 6-hydroxyhexyl ester to6-hydroxyhexanoate and/or 1,6-hexanediol. In some embodiments, apolypeptide having esterase activity enzymatically converts6-hydroxyhexanoic acid hexyl ester, 6-hydroxyhexanoic acid6-hydroxyhexyl ester, or hexanoic acid 6-hydroxyhexyl ester to6-hydroxyhexanoate and/or 1,6-hexanediol.

In some embodiments, the method further includes enzymaticallyconverting 1,6-hexanediol to 6-hydroxyhexanal. In some embodiments, apolypeptide having alcohol dehydrogenase activity enzymatically converts1,6-hexanediol to 6-hydroxyhexanal.

In some embodiments, the method further includes enzymaticallyconverting 6-hydroxyhexanal to 6-hydroxyhexanoate. In some embodiments,a polypeptide having aldehyde dehydrogenase activity enzymaticallyconverts 6-hydroxyhexanal to 6-hydroxyhexanoate.

In some embodiments, the method further includes enzymaticallyconverting 6-hydroxyhexanoate methyl ester to 6-hydroxyhexanoate. Insome embodiments, a polypeptide having demethylase or esterase activityenzymatically converts 6-hydroxyhexanoate methyl ester to6-hydroxyhexanoate.

In some embodiments, the method further includes enzymaticallyconverting 6-hydroxyhexanoate to a product selected from the groupconsisting of adipic acid, adipate semialdehyde, 6-aminohexanoate,hexamethylenediamine, and 1,6-hexanediol.

In some embodiments, the method includes enzymatically converting6-hydroxyhexanoate to adipate semialdehyde using a polypeptide havingalcohol dehydrogenase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, or a polypeptide having monooxygenase activity.

In some embodiments, the method further the enzymatically convertingadipate semialdehyde to adipic acid using a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide having aldehydedehydrogenase activity, or a polypeptide having monooxygenase activity.

In some embodiments, the method further includes enzymaticallyconverting adipate semialdehyde to 6-aminohexanoate. In someembodiments, a polypeptide having ω-transaminase activity enzymaticallyconverts adipate semialdehyde to 6-aminohexanoate.

In some embodiments, the method further includes enzymaticallyconverting 6-aminohexanoate to hexamethylenediamine. In someembodiments, the method further includes enzymatically convertingadipate semialdehyde to hexamethylenediamine. In some embodiments,adipate semialdehyde or 6-aminohexanoate is enzymatically converted tohexamethylenediamine using a polypeptide having carboxylate reductaseactivity and/or a polypeptide having ω-transaminase activity andoptionally one or more of a polypeptide having N-acetyl transferaseactivity, a polypeptide having acetylputrescine deacetylase activity,and a polypeptide having alcohol dehydrogenase activity.

In some embodiments, said method comprises enzymatically converting6-hydroxyhexanoate to 1,6-hexanediol using a polypeptide havingcarboxylate reductase activity and a polypeptide having alcoholdehydrogenase activity.

In some embodiments, said method further comprises enzymaticallyconverting 1,6-hexanediol to hexamethylenediamine. In some embodiments,a polypeptide having alcohol dehydrogenase activity and a polypeptidehaving ω-transaminase activity enzymatically converts 1,6-hexanediolhexamethylenediamine.

In some embodiments, a polypeptide having carboxylate reductaseactivity, a polypeptide having ω-transaminase activity, and apolypeptide having alcohol dehydrogenase activity enzymatically converts6-hydroxyhexanoate to hexamethylenediamine.

In some embodiments, the polypeptide having ω-transaminase activity hasat least 70% sequence identity to any one of the amino acid sequencesset forth in SEQ ID NO. 7-12.

In some embodiments, hexanoyl-CoA is produced from acetyl-CoA via twocycles of CoA-dependent carbon chain elongation. In some embodiments,each of said two cycles of CoA-dependent carbon chain elongationcomprises using a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity to form hexanoyl-CoA from acetyl-CoA.

Enzymes Converting Hexanoate or Hexanoyl-CoA to 6-Hydroxyhexanoate

As depicted in FIG. 7, hexanoate methyl ester can be formed fromhexanoate using a polypeptide having fatty acid O-methyltransferaseactivity, such as the polypeptide having fatty acid O-methyltransferaseactivity classified, for example, under EC 2.1.1.15. For example, thepolypeptide having fatty acid O-methyltransferase activity can beobtained from Mycobacterium marinum (GenBank Accession No. ACC41782.1.SEQ ID NO:22); Mycobacterium smegmatis (see GenBank Accession No.ABK73223.1, SEQ ID NO: 23), or Pseudomonas putida (see GenBank AccessionNo. CAA39234.1, SEQ ID NO: 24).

Hexanoate methyl ester can be converted to 6-hydroxyhexanoate methylester using a polypeptide having monooxygenase activity classified, forexample, under EC 1.14.14.- or EC 1.14.15.-(1,3) For example, apolypeptide having monooxygenase activity can be, for example, from theCYP153A family (SEQ ID NO:13-15), the CYP52A3 family (See GenbankAccession No: BAA00371.1, SEQ ID NO: 28) or the alkB family such as thegene product of alkBGT from Pseudomonas putida (See Genbank AccessionNo. CAB51047.1, SEQ ID NO: 27). See, FIG. 7.

6-hydroxyhexanoate methyl ester can be converted to 6-hydroxyhexanoateusing a polypeptide having demethylase activity classified, for example,under EC 2.1.1.- such as the gene product of ligM (see GenBank AccessionNo. BAD61059.1; SEQ ID NO:30) or desA (GenBank Accession No. BAC79257.1;SEQ ID NO:31) or using a polypeptide having esterase activityclassified, for example under EC 3.1.1.- such as the gene product ofEstC (see GenBank Accession No. AAC60471.2, SEQ ID NO: 26).

As depicted in FIG. 7, hexanoyl-CoA can be converted to hexanoic acidhexyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.-(84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 25).

Hexanoic acid hexyl ester can be converted to 6-hydroxyhexanoic acidhexyl ester and/or 6-hydroxyhexanoic acid 6-hydroxyhexyl ester using apolypeptide having monooxygenase activity classified, for example, underEC 1.14.14.- or EC 1.14.15.-(1,3) For example, a polypeptide havingmonooxygenase activity can be, for example, from the CYP153A family, theCYP52A3 family (Genbank Accession No: BAA00371.1, SEQ ID NO: 28) or thealkB family such as the gene product of alkBGT from Pseudomonas putida(Genbank Accession No. CAB51047.1, SEQ ID NO: 27). See, FIG. 7.

6-hydroxyhexanoic acid hexyl ester and 6-hydroxyhexanoic acid6-hydroxyhexyl ester can be converted to 6-hydroxyhexanoate using apolypeptide having esterase activity classified, for example, under EC3.1.1.-(1,6) such as the gene product of EstC (see GenBank Accession No.AAC60471.2, SEQ ID NO: 26).

For example, the monooxygenase CYP153A family classified, for example,under EC 1.14.15.-(1,3) is soluble and has regio-specificity forterminal hydroxylation, accepting medium chain length substrates (see,e.g., Koch et al., Appl. Environ. Microbiol., 2009, 75(2), 337-344;Funhoff et al., 2006, J. Bacteriol., 188(44): 5220-5227; Van Beilen &Funhoff, Current Opinion in Biotechnology, 2005, 16, 308-314; Nieder andShapiro, J. Bacteriol., 1975, 122(1), 93-98). Although non-terminalhydroxylation is observed in vitro for CYP153A, in vivo only1-hydroxylation occurs (see, Funhoff et al., 2006, supra).

The substrate specificity and activity of terminal monooxygenases hasbeen broadened via successfully, reducing the chain length specificityof CYP153A to below C8 (Koch et al., 2009, supra).

In some embodiments, hexanoate can be enzymatically formed fromhexanoyl-CoA using a polypeptide having thioesterase activity classifiedunder EC 3.1.2.-, such as the gene product of YciA, tesB (GenBankAccession No. AAA24665.1, SEQ ID NO: 1), Acot13 (Cantu et al., ProteinScience, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008,47(9):2789-2796; Naggert et al., J. Biol. Chem., 1991,266(17):11044-11050), the acyl-[acp] thioesterase from a Lactobacillusbrevis (GenBank Accession No. ABJ63754.1, SEQ ID NO:32), or aLactobacillus plantarum (GenBank Accession No. CCC78182.1, SEQ IDNO:33). Such acyl-[acp] thioesterases have C6-C8 chain lengthspecificity (see, for example, Jing et al., 2011, BMC Biochemistry,12(44)). See, FIG. 3.

In some embodiments, hexanoate can be enzymatically formed fromhexanoyl-CoA using a polypeptide having butanal dehydrogenase activityclassified, for example, under EC 1.2.1.- such as EC 1.2.1.10 or EC1.2.1.57 (see, e.g., GenBank Accession No. AAD39015, SEQ ID NO:29 orGenBank Accession No. ABJ64680.1) such as the gene product of PduP orPduB and an aldehyde dehydrogenase classified, for example, under EC1.2.1.- (e.g., EC 1.2.1.3 or EC 1.2.1.4) (see, Ho & Weiner, J.Bacteriol., 2005, 187(3):1067-1073). See, FIG. 3.

Enzymes Generating Hexanoyl-CoA for Conversion to a C6 Building Block

As depicted in FIG. 1 and FIG. 2, hexanoyl-CoA can be formed fromacetyl-CoA via two cycles of CoA-dependent carbon chain elongation usingeither NADH or NADPH dependent enzymes.

In some embodiments, a CoA-dependent carbon chain elongation cyclecomprises using a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity and a polypeptide having trans-2-enoyl-CoA reductaseactivity. A polypeptide having β-ketothiolase activity can convertacetyl-CoA to 3-oxobutanoyl-CoA and can convert butanoyl-CoA to3-oxohexanoyl-CoA. A polypeptide having acetyl-CoA carboxylase activitycan convert acetyl-CoA to malonyl-CoA. A polypeptide havingacetoacetyl-CoA synthase activity can convert malonyl-CoA toacetoacetyl-CoA. A polypeptide having 3-hydroxybutyryl-CoA dehydrogenaseactivity can convert 3-oxobutanoyl-CoA to 3-hydroxybutanoyl CoA. Apolypeptide having 3-oxoacyl-CoA reductase/3-hydroxyacyl-CoAdehydrogenase activity can convert 3-oxohexanoyl-CoA to3-hydroxyhexanoyl-CoA. A polypeptide having enoyl-CoA hydratase activitycan convert 3-hydroxybutanoyl-CoA to but-2-enoyl-CoA and can convert3-hydroxyhexanoyl-CoA to hex-2-enoyl-CoA. A polypeptide havingtrans-2-enoyl-CoA reductase activity can convert but-2-enoyl-CoA tobutanoyl-CoA and can convert hex-2-enoyl-CoA to hexanoyl-CoA. See FIGS.1 and 2.

In some embodiments, a polypeptide having β-ketothiolase activity can beclassified under EC 2.3.1.16, such as the gene product of bktB (See,e.g., Genbank Accession No. AAC38322.1). The polypeptide havingβ-ketothiolase activity encoded by bktB from Cupriavidus necator acceptsbutanoyl-CoA as substrates. When butanoyl-CoA is the substrate, theCoA-activated C6 aliphatic backbone (3-oxohexanoyl-CoA) is produced(see, e.g., Haywood et al., FEMS Microbiology Letters, 1988, 52:91-96;Slater et al., J. Bacteriol., 1998, 180(8):1979-1987). The polypeptidehaving β-ketothiolase activity encoded by paaJ (See, e.g., GenbankAccession No. AAC74479.1), catF and pcaF can be classified under, forexample, EC 2.3.1.174. The polypeptide having β-ketothiolase activityencoded by paaJ condenses acetyl-CoA and succinyl-CoA to 3-oxoadipyl-CoA(see, for example, Fuchs et al., 2011, Nature Reviews Microbiology, 9,803-816; Gael et al., 2002, J. Bacteriol., 184(1), 216-223) See FIGS. 1and 2.

In some embodiments, a polypeptide having acetyl-CoA carboxylaseactivity can be classified, for example, under EC 6.4.1.2. In someembodiments, a polypeptide having β-ketoacyl-[acp] synthase activity canbe classified, for example, under 2.3.1.180 such as the gene product ofFabH from Staphylococcus aereus (Qiu et al., 2005, Protein Science, 14:2087-2094). See FIGS. 1 and 2.

In some embodiments, a polypeptide having 3-hydroxyacyl-CoAdehydrogenase activity or a polypeptide having 3-oxoacyl-CoAdehydrogenase activity can be classified under EC 1.1.1.-. For example,the polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity can beclassified under EC 1.1.1.35, such as the gene product of fadB (FIG. 1);classified under EC 1.1.1.157, such as the gene product of hbd (can bereferred to as a 3-hydroxybutyryl-CoA dehydrogenase) (FIG. 1); orclassified under EC 1.1.1.36, such as the acetoacetyl-CoA reductase geneproduct of phaB (Liu & Chen, Appl. Microbiol. Biotechnol., 2007,76(5):1153-1159; Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915; Budde et al., J. Bacteriol., 2010, 192(20):5319-5328)(FIG. 2).

In some embodiments, a polypeptide having 3-oxoacyl-CoA reductaseactivity can be classified under EC 1.1.1.100, such as the gene productoffabG (Budde et al., J. Bacteriol., 2010, 192(20):5319-5328; Nomura etal., Appl. Environ. Microbiol., 2005, 71(8):4297-4306). FIG. 2.

In some embodiments, a polypeptide having enoyl-CoA hydratase activitycan be classified under EC 4.2.1.17, such as the gene product of crt(Genbank Accession No. AAA95967.1) (FIG. 1), or classified under EC4.2.1.119, such as the gene product of phaJ (Genbank Accession No.BAA21816.1) (FIG. 2) (Shen et al., 2011, supra; Fukui et al., J.Bacteriol., 1998, 180(3):667-673).

In some embodiments, a polypeptide having trans-2-enoyl-CoA reductaseactivity can be classified under EC 1.3.1.38 (FIG. 2), EC 1.3.1.8 (FIG.2), or EC 1.3.1.44 (FIG. 1), such as the gene product of ter (GenbankAccession No. AAW66853.1) (Nishimaki et al., J. Biochem., 1984,95:1315-1321; Shen et al., 2011, supra) or tdter (Genbank Accession No.AAS11092.1) (Bond-Watts et al., Biochemistry, 2012, 51:6827-6837).

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of aC6 Building Block

As depicted in FIGS. 4, 5, and 7, a terminal carboxyl group can beenzymatically formed using a polypeptide having thioesterase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide havingmonooxygenase activity, a polypeptide having esterase activity or apolypeptide having demethylase activity.

In some embodiments, the first terminal carboxyl group is enzymaticallyformed by a polypeptide having syringate O-demethylase activityclassified under EC 2.1.1.- such as the gene products of ligM (seeGenBank Accession No. BAD61059.1; SEQ ID NO:30) or desA (GenBankAccession No. BAC79257.1; SEQ ID NO:31) or a polypeptide having esteraseactivity classified under EC 3.1.1.- such as the gene product of EstC(see, e.g., GenBank Accession No. AAC60471.2, SEQ ID NO: 26). See, e.g.,FIG. 7.

In some embodiments, the first terminal carboxyl group is enzymaticallyformed by a polypeptide having aldehyde dehydrogenase activityclassified, for example, under EC 1.2.1.3 or EC 1.2.1.4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of adipic acid is enzymatically formed by a polypeptide havingaldehyde dehydrogenase activity classified, for example, under EC1.2.1.3 (see, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81,185-192). See FIG. 4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of adipic acid is enzymatically formed by a polypeptide havingdehydrogenase activity classified under EC 1.2.1.- such as a polypeptidehaving 5-oxopentanoate dehydrogenase activity (e.g., the gene product ofCpnE), a polypeptide having 6-oxohexanoate dehydrogenase activity (e.g.,the gene product of ChnE from Acinetobacter sp.), a polypeptide having7-oxoheptanoate dehydrogenase activity (e.g., the gene product of ThnGfrom Sphingomonas macrogolitabida) (Iwaki et al., Appl. Environ.Microbiol., 1999, 65(11), 5158-5162; López-Sánchez et al., Appl.Environ. Microbiol., 2010, 76(1), 110-118). For example, a polypeptidehaving 5-oxopentanoate dehydrogenase activity can be classified under EC1.2.1.20. For example, a polypeptide having 6-oxohexanoate dehydrogenaseactivity can be classified under EC 1.2.1.63. For example, a polypeptidehaving 7-oxoheptanoate dehydrogenase activity can be classified under EC1.2.1.-. See, FIG. 4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of adipic acid is enzymatically formed by a polypeptide havingmonooxygenase activity in the cytochrome P450 family such as CYP4F3B(see, e.g., Sanders et al., J. Lipid Research, 2005, 46(5):1001-1008;Sanders et al., The FASEB Journal, 2008, 22(6):2064-2071). See, FIG. 4.

The utility of ω-oxidation in introducing carboxyl groups into alkaneshas been demonstrated in the yeast Candida tropicalis, leading to thesynthesis of adipic acid (Okuhara et al., Agr. Biol. Chem., 1971, 35(9),1376-1380).

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of a C6Building Block

As depicted in FIG. 5 and FIG. 6, terminal amine groups can beenzymatically formed using a polypeptide having ω-transaminase activityor a polypeptide having deacetylase activity.

In some embodiments, the first terminal amine group leading to thesynthesis of 6-aminohexanoic acid, 6-aminohexanal, or 6-aminohexanol isenzymatically formed by a ω-transaminase classified, for example, underEC 2.6.1.- such as EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48,or EC 2.6.1.82 such as that obtained from Chromobacterium violaceum(Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa(Genbank Accession No. AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae(Genbank Accession No. AAY39893.1, SEQ ID NO: 9), Rhodobactersphaeroides (Genbank Accession No. ABA81135.1, SEQ ID NO: 10), VibrioFluvialis (Genbank Accession No. AAA57874.1, SEQ ID NO: 12),Streptomyces griseus, or Clostridium viride. An additional polypeptidehaving ω-transaminase activity that can be used in the methods and hostsdescribed herein is from Escherichia coli (Genbank Accession No.AAA57874.1, SEQ ID NO: 11). Some of the polypeptides havingω-transaminase activity classified, for example, under EC 2.6.1.29 or EC2.6.1.82 are polypeptides having diamine ω-transaminase activity (e.g.,SEQ ID NO:11). See, e.g., FIGS. 5 and 6.

The reversible polypeptide having ω-transaminase activity fromChromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO:7) has demonstrated analogous activity accepting 6-aminohexanoic acid asamino donor, thus forming the first terminal amine group in adipatesemialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007,41, 628-637).

The reversible polypeptide having 4-aminobutyrate:2-oxoglutaratetransaminase activity from Streptomyces griseus has demonstratedanalogous activity for the conversion of 6-aminohexanoate to adipatesemialdehyde (Yonaha et al., Eur. J. Biochem., 1985, 146, 101-106).

The reversible polypeptide having 5-aminovalerate transaminase activityfrom Clostridium viride has demonstrated analogous activity for theconversion of 6-aminohexanoate to adipate semialdehyde (Barker et al.,J. Biol. Chem., 1987, 262(19), 8994-9003).

In some embodiments, the second terminal amine group leading to thesynthesis of hexamethylenediamine is enzymatically formed by apolypeptide having diamine transaminase activity. For example, thesecond terminal amino group can be enzymatically formed by a polypeptidehaving diamine transaminase activity classified, for example, under EC2.6.1.-, e.g., EC 2.6.1.29 or classified, for example, under EC2.6.1.82, such as the gene product of YgjG from E. coli (GenbankAccession No. AAA57874.1, SEQ ID NO: 12). See, FIG. 6.

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine(Samsonova et al., BMC Microbiology, 2003, 3:2).

The polypeptide having diamine transaminase activity from E. coli strainB has demonstrated activity for 1,7 diaminoheptane (Kim, The Journal ofChemistry, 1964, 239(3), 783-786).

In some embodiments, the second terminal amine group leading to thesynthesis of hexamethylenediamine is enzymatically formed by apolypeptide having deacetylase activity classified, for example, underEC 3.5.1.62 such as a polypeptide having acetylputrescine deacetylaseactivity. The polypeptide having acetylputrescine deacetylase activityfrom Micrococcus luteus K-11 accepts a broad range of carbon chainlength substrates, such as acetylputrescine, acetylcadaverine andN⁸-acetylspermidine (see, for example, Suzuki et al., 1986, BBA—GeneralSubjects, 882(1):140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of aC6 Building Block

As depicted in FIG. 8, a terminal hydroxyl group can be enzymaticallyforming using a polypeptide having alcohol dehydrogenase activity. Forexample, the second terminal hydroxyl group leading to the synthesis of1,6 hexanediol can be enzymatically formed by a polypeptide havingalcohol dehydrogenase activity classified under EC 1.1.1.- (e.g., EC1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184). A first terminal hydroxylgroup can be enzymatically formed with a polypeptide having monoxygenaseactivity as discussed above with respect to the formation of6-hydroxyhexanoate methyl ester in FIG. 7.

As depicted in FIG. 8, hexanoyl-CoA can be converted to hexanoic acidhexyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.-(84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 25).

Hexanoic acid hexyl ester can be converted to 6-hydroxyhexanoic acid6-hydroxyhexyl ester using a polypeptide having monooxygenase activityclassified, for example, under EC 1.14.14.- or EC 1.14.15.-(1,3).Hexanoic acid hexyl ester can be converted to hexanoic acid6-hydroxyhexyl ester using a polypeptide having monooxygenase activityclassified, for example, under EC 1.14.14.- or EC 1.14.15.-(1,3). Forexample, a polypeptide having monooxygenase activity can be, forexample, from the CYP153A family, the CYP52A3 family or the alkB familysuch as the gene product of alkBGT from Pseudomonas putida. See, FIG. 7.

Hexanoic acid 6-hydroxyhexyl ester and 6-hydroxyhexanoic acid6-hydroxyhexyl can be converted to 1,6-hexanediol using a polypeptidehaving esterase activity classified, for example, under EC 3.1.1.-(e.g., EC 3.1.1.1 or EC 3.1.1.6) such as the gene product of EstC (seeGenBank Accession No. AAC60471.2, SEQ ID NO: 26).

Biochemical Pathways

Pathways to Hexanoyl-CoA as Central Precursor to C6 Building Blocks

In some embodiments, hexanoyl-CoA is synthesized from acetyl-CoA byconversion of acetyl-CoA to 3-oxobutanoyl-CoA by a polypeptide havingβ-ketothiolase activity classified, for example, under EC 2.3.1.9, suchas the gene product of phaA or atoB; followed by conversion of3-oxobutanoyl-CoA to (S) 3-hydroxybutanoyl-CoA by a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity classified, for example, underEC 1.1.1.35, such as the gene product of fadB or classified, forexample, under EC 1.1.1.157 such as the gene product of hbd; followed byconversion of (S) 3-hydroxybutanoyl-CoA to but-2-enoyl-CoA by apolypeptide having enoyl-CoA hydratase activity classified, for example,under EC 4.2.1.17 such as the gene product of crt (Genbank Accession No.AAA95967.1); followed by conversion of but-2-enoyl-CoA to butanoyl-CoAby a polypeptide having trans-2-enoyl-CoA reductase activity classified,for example, under EC 1.3.1.44 such as the gene product of ter (GenbankAccession No. AAW66853.1) or tdter (Genbank Accession No. AAS11092.1);followed by conversion of butanoyl-CoA to 3-oxo-hexanoyl-CoA by apolypeptide having β-ketothiolase activity classified, for example,under EC 2.3.1.16 such as the gene product of bktB (Genbank AccessionNo. AAC38322.1) or classified, for example, under EC 2.3.1.174 such asthe gene product of paaJ (Genbank Accession No. AAC74479.1); followed byconversion of 3-oxo-hexanoyl-CoA to (S) 3-hydroxyhexanoyl-CoA by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.35 such as the gene product of fadB or by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.157 such as the gene product of hbd;followed by conversion of (S) 3-hydroxyhexanoyl-CoA to hex-2-enoyl-CoAby a polypeptide having enoyl-CoA hydratase activity classified, forexample, under EC 4.2.1.17 such as the gene product of crt (GenbankAccession No. AAA95967.1); followed by conversion of hex-2-enoyl-CoA tohexanoyl-CoA by a polypeptide having trans-2-enoyl-CoA reductaseactivity classified, for example, under EC 1.3.1.44 such as the geneproduct of ter (Genbank Accession No. AAW66853.1) or tdter (GenbankAccession No. AAS11092.1). See FIG. 1.

In some embodiments, hexanoyl-CoA is synthesized from the centralmetabolite, acetyl-CoA, by conversion of acetyl-CoA to 3-oxobutanoyl-CoAby a polypeptide having fl-ketothiolase activity classified, forexample, under EC 2.3.1.9, such as the gene product of phaA or atoB;followed by conversion of 3-oxobutanoyl-CoA to (R) 3-hydroxybutanoyl-CoAby a polypeptide having 3-oxoacyl-CoA reductase activity classified, forexample, under EC 1.1.1.100, such as the gene product of fadG or by apolypeptide having acetoacetyl-CoA reductase activity classified, forexample, under EC 1.1.1.36 such as the gene product of phaB; followed byconversion of (R) 3-hydroxybutanoyl-CoA to but-2-enoyl-CoA by apolypeptide having enoyl-CoA hydratase activity classified, for example,under EC 4.2.1.119 such as the gene product of phaJ (Genbank AccessionNo. BAA21816.1); followed by conversion of but-2enoyl-CoA tobutanoyl-CoA by a polypeptide having trans-2-enoyl-CoA reductaseactivity classified, for example, under EC 1.3.1.38 or a polypeptidehaving acyl-CoA dehydrogenase activity classified, for example, under EC1.3.1.8; followed by conversion of butanoyl-CoA to 3-oxo-hexanoyl-CoA bya polypeptide having β-ketothiolase activity classified, for example,under EC 2.3.1.16 such as the gene product of bktB (Genbank AccessionNo. AAC38322.1) or classified, for example, under EC 2.3.1.174 such asthe gene product of paaJ (Genbank Accession No. AAC74479.1); followed byconversion of 3-oxo-hexanoyl-CoA to (R) 3-hydroxyhexanoyl-CoA by apolypeptide having 3-oxoacyl-CoA reductase activity classified, forexample, under EC 1.1.1.100 such as the gene product of fabG; followedby conversion of (R) 3-hydroxyhexanoyl-CoA to hex-2-enoyl-CoA by apolypeptide having enoyl-CoA hydratase activity classified, for example,under EC 4.2.1.119 such as the gene product of phaJ (Genbank AccessionNo. BAA21816.1); followed by conversion of hex-2-enoyl-CoA tohexanoyl-CoA by a polypeptide having trans-2-enoyl-CoA reductaseactivity classified, for example, under EC 1.3.1.38 or a polypeptidehaving acyl-CoA dehydrogenase activity classified, for example, under EC1.3.1.8. See FIG. 2.

In some embodiments, 3-oxobutanoyl-CoA can be synthesized fromacetyl-CoA. A polypeptide having acetyl-CoA carboxylase activityclassified, for example, under EC 6.4.1.2 can be used to convertacetyl-CoA to malonyl-CoA, which can be converted to 3-oxobutanoyl-CoAusing a polypeptide having acetoacetyl-CoA synthase activity classified,for example, under EC 2.3.1.194 such as the gene product of fabH. See,FIG. 1 and FIG. 2.

Pathways Using Hexanoyl-CoA to Produce the Central Precursor Hexanoate

In some embodiments, hexanoate is synthesized from hexanoyl-CoA byconversion of hexanoyl-CoA to hexanoate by a polypeptide havingthioesterase activity classified, for example, under EC 3.1.2.- such asthe gene product of YciA, tesB, Acot13, a Lactobacillus brevisacyl-[acp] thioesterase (GenBank Accession No. ABJ63754.1, SEQ ID NO:32)or a Lactobacillus plantarum acyl-[acp] thioesterase (GenBank AccessionNo. CCC78182.1, SEQ ID NO:33). See, FIG. 3.

In some embodiments, hexanoyl-CoA is converted to hexanal by apolypeptide having butanal dehydrogenase activity classified, forexample, under EC 1.2.1.57 (see, e.g, GenBank Accession No. AAD39015,SEQ ID NO:29) or EC 1.2.1.10 or the gene products of PduP or PduB;followed by conversion of hexanal to hexanoate by a polypeptide havingaldehyde dehydrogenase activity classified, for example, under EC1.2.1.4 or EC 1.2.1.3. See FIG. 3.

The conversion of hexanoyl-CoA to hexanal has been demonstrated usingboth NADH and NADPH as co-factors (see Palosaari and Rogers, J.Bacteriol., 1988, 170(7): 2971-2976).

Pathways Using Hexanoyl-CoA to Produce the Central Precursor Hexanol

In some embodiments, hexanoate is synthesized from hexanoyl-CoA byconversion of hexanoyl-CoA to hexanoate by a polypeptide havingthioesterase activity classified, for example, under EC 3.1.2.- such asthe gene product of YciA, tesB or Acot13, a Lactobacillus brevisacyl-[acp] thioesterase (GenBank Accession No. ABJ63754.1, SEQ ID NO:32)or a Lactobacillus plantarum acyl-[acp] thioesterase (GenBank AccessionNo. CCC78182.1, SEQ ID NO:33); followed by conversion of hexanoate tohexanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6, such as the gene product ofcar enhanced by the gene product of sfp; followed by conversion ofhexanal to hexanol by a polypeptide having alcohol dehydrogenaseactivity classified, for example, under EC 1.1.1.- such as EC 1.1.1.1,EC 1.1.1.2, EC 1.1.1.21, or EC 1.11184) such as the gene product ofYMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E. coli,GenBank Accession No. AAA69178.1) (see, e.g., Liu et al., Microbiology,2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1),163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257)or the protein having GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus). See, FIG. 3.

In some embodiments, hexanoyl-CoA is converted to hexanal by apolypeptide having butanal dehydrogenase activity classified, forexample, under EC 1.2.1.57 (see, e.g, GenBank Accession No. BAD61059.1,SEQ ID NO:30); followed by conversion of hexanal to hexanol by apolypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21,or EC 1.1.1.184) such as the gene product of YMR318C (Genbank AccessionNo. CAA90836.1) or YqhD (from E. coli, GenBank Accession No. AAA69178.1)(see, e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy etal., 2002, Biochem J., 361(Pt 1), 163-172; or Jarboe, 2011, Appl.Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBankAccession No. CAA81612.1 (from Geobacillus stearothermophilus). See,FIG. 3.

Pathways Using Hexanoate or Hexanoyl-CoA as Central Precursor to6-Hydroxyhexanoate

In some embodiments, 6-hydroxyhexanoate is synthesized from the centralprecursor, hexanoate, by conversion of hexanoate to hexanoate methylester using a polypeptide having fatty acid O-methyltransferase activityclassified, for example, under EC 2.1.1.15 (e.g., the fatty acidO-methyltransferase from Mycobacterium marinum (GenBank Accession No.ACC41782.1. SEQ ID NO:22), Mycobacterium smegmatis (see GenBankAccession No. ABK73223.1, SEQ ID NO: 23), or Pseudomonas putida (seeGenBank Accession No. CAA39234.1, SEQ ID NO: 24); followed by conversionto 6-hydroxyhexanoate methyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.-(1,3) such as a polypeptide having monooxygenase activity inthe CYP153A, the CYP52A3 family or alkB family; followed by conversionto 6-hydroxyhexanoate using a polypeptide having syringate O-demethylaseactivity classified under EC 2.1.1.- such as the gene products of ligM(see GenBank Accession No. BAD61059.1; SEQ ID NO:30) or desA (GenBankAccession No. BAC79257.1; SEQ ID NO:31) or using a polypeptide havingesterase activity classified under EC 3.1.1.- such as the gene productof EstC (see GenBank Accession No. AAC60471.2, SEQ ID NO: 26) (Kim etal., 1994, Biosci. Biotech. Biochem, 58(1), 111-116).

In some embodiments, hexanoate can be enzymatically converted to6-hydroxyhexanoate by a polypeptide having monooxygenase activity(classified, for example, under EC 1.14.14.- or EC 1.14.15.- (e.g., EC1.14.15.1 or EC 1.14.15.3 such as a monooxygenase in the CYP153A,theCYP52A3 family or/and the alkB family).

In some embodiments, hexanoyl-CoA can be converted to hexanoic acidhexyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.-(84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 25);followed by conversion to 6-hydroxyhexanoic acid hexyl ester and/or6-hydroxyhexanoic acid 6-hydroxyhexyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.-(1,3). For example, a polypeptide having monooxygenase activitycan be, for example, from the CYP153A family, the CYP52A3 family(Genbank Accession No: BAA00371.1, SEQ ID NO: 28) or the alkB familysuch as the gene product of alkBGT from Pseudomonas putida (GenbankAccession No. CAB51047.1, SEQ ID NO: 27); followed by conversion of6-hydroxyhexanoic acid hexyl ester and/or 6-hydroxyhexanoic acid6-hydroxyhexyl to 6-hydroxyhexanoate using a polypeptide having esteraseactivity classified, for example, under EC 3.1.1.-(1,6) such as the geneproduct of EstC (see GenBank Accession No. AAC60471.2, SEQ ID NO: 26)(Kim et al., 1994, Biosci. Biotech. Biochem, 58(1), 111-116). See FIG.7.

Pathways Using 6-Hydroxyhexanoate as Central Precursor to Adipate

Adipate semialdehyde can be synthesized by enzymatically converting6-hydroxyhexanoate to adipate semialdehyde using a polypeptide havingalcohol dehydrogenase activity classified, for example, under EC 1.1.1.-such as the gene product of YMR318C (classified, for example, under EC1.1.1.2, see Genbank Accession No. CAA90836.1) (Larroy et al., 2002,Biochem J., 361 (Pt 1), 163-172), cpnD (Iwaki et al., 2002, Appl.Environ. Microbiol., 68(11):5671-5684) or gbd, a polypeptide having6-hydroxyhexanoate dehydrogenase activity classified, for example, underEC 1.1.1.258 such as the gene product of ChnD (Iwaki et al., Appl.Environ. Microbiol., 1999, 65(11):5158-5162), or a polypeptide havingcytochrome P450 activity (Sanders et al., J. Lipid Research, 2005,46(5), 1001-1008; Sanders et al., The FASEB Journal, 2008, 22(6),2064-2071). See, FIG. 4. The polypeptide having alcohol dehydrogenaseactivity encoded by YMR318C has broad substrate specificity, includingthe oxidation of C6 alcohols.

Adipate semialdehyde can be enzymatically converted to adipic acid usinga polypeptide having aldehyde dehydrogenase activity classified, forexample, under EC 1.2.1.-(3, 16, 20, 63, 79) such as a polypeptidehaving 7-oxoheptanoate dehydrogenase activity (e.g., the gene product ofThnG), a polypeptide having 6-oxohexanoate dehydrogenase activity (e.g.,the gene product of ChnE). See FIG. 4.

Pathway Using 6-Hydroxyhexanoate as Central Precursor to6-Aminohexanoate

In some embodiments, 6-aminohexanoate is synthesized from6-hydroxyhexanoate by conversion of 6-hydroxyhexanoate to adipatesemialdehyde using a polypeptide having alcohol dehydrogenase activityclassified, for example, under EC 1.1.1.- such as the gene product ofYMR318C (classified, for example, under EC 1.1.1.2, see GenbankAccession No. CAA90836.1) (Larroy et al., 2002, Biochem J., 361(Pt 1),163-172), cpnD (Iwaki et al., 2002, Appl. Environ. Microbiol.,68(11):5671-5684), or gbd, or a 6-hydroxyhexanoate dehydrogenaseclassified, for example, under EC 1.1.1.258 such as the gene product ofChnD (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11):5158-5162);followed by conversion to 6-aminohexanoate by a polypeptide havingω-transaminase activity classified, for example, under EC 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.82 such as from aChromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ IDNO: 7), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1,SEQ ID NO: 8), a Pseudomonas syringae (see Genbank Accession No.AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides (see GenbankAccession No. ABA81135.1, SEQ ID NO: 10), or a Vibrio fluvialis (seeGenbank Accession No. AEA39183.1, SEQ ID NO: 12). See FIG. 5.

Pathway Using 6-Aminohexanoate, 6-Hydroxyhexanoate, or AdipateSemialdehyde as Central Precursor to Hexamethylenediamine

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor 6-aminohexanoate by conversion of 6-aminohexanoate to6-aminohexanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 such as the gene product ofcar in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene products of GriC and GriD from Streptomyces griseus;followed by conversion of 6-aminohexanal to hexamethylenediamine by apolypeptide having ω-transaminase activity classified, for example,under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as from a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQID NO: 10), an Escherichia coli (see Genbank Accession No. AAA57874.1,SEQ ID NO: 11), or a Vibrio fluvialis (see Genbank Accession No.AEA39183.1, SEQ ID NO: 12). See FIG. 6.

The polypeptide having carboxylate reductase activity encoded by thegene product of car and enhancer npt or sfp has broad substratespecificity, including terminal difunctional C4 and C5 carboxylic acids(Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42,130-137).

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor 6-hydroxyhexanoate (which can be produced as describedin FIG. 7), by conversion of 6-hydroxyhexanoate to 6-hydroxyhexanal by apolypeptide having carboxylate reductase activity classified, forexample, under EC 1.2.99.6 such as from Mycobacterium marinum (GenbankAccession No. ACC40567.1, SEQ ID NO: 2), Mycobacterium smegmatis(Genbank Accession No. ABK71854.1, SEQ ID NO: 3), Segniliparus rugosus(Genbank Accession No. EFV11917.1, SEQ ID NO: 4), Mycobacteriummassiliense (Genbank Accession No. EIV11143.1, SEQ ID NO: 5), orSegniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 6)in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:20) genefrom Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ IDNO:21) gene from Nocardia), or the gene products of GriC and GriD fromStreptomyces griseus; followed by conversion of 6-oxohexanol to6-aminohexanol by a polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 7), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:8), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1,SEQ ID NO: 10), an Escherichia coli (see Genbank Accession No.AAA57874.1, SEQ ID NO: 11), or a Vibrio fluvialis (see Genbank AccessionNo. AEA39183.1, SEQ ID NO: 12); followed by conversion to 6-aminohexanalby a polypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, orEC 1.1.1.184) such as the gene product of YMR318C (Genbank Accession No.CAA90836.1) or YqhD (from E. coli, GenBank Accession No. AAA69178.1)(Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002,Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol.Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No.CAA81612.1; followed by conversion to hexamethylenediamine by apolypeptide having ω-transaminase activity classified, for example,under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as from a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQID NO: 10), an Escherichia coli (see Genbank Accession No. AAA57874.1,SEQ ID NO: 11), or a Vibrio fluvialis (see Genbank Accession No.AEA39183.1, SEQ ID NO: 12). See FIG. 6.

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor 6-aminohexanoate by conversion of 6-aminohexanoate toN6-acetyl-6-aminohexanoate by a polypeptide having N-acetyltransferaseactivity such as a polypeptide having lysine N-acetyltransferaseactivity classified, for example, under EC 2.3.1.32; followed byconversion to N6-acetyl-6-aminohexanal by a polypeptide havingcarboxylate reductase activity classified, for example, under EC1.2.99.6 such as from Segniliparus rugosus (Genbank Accession No.EFV11917.1, SEQ ID NO: 4), Mycobacterium massiliense (Genbank AccessionNo. EIV11143.1, SEQ ID NO: 5), or Segniliparus rotundus (GenbankAccession No. ADG98140.1, SEQ ID NO: 6) in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp (GenbankAccession No. CAA44858.1, SEQ ID NO:20) gene from Bacillus subtilis ornpt (Genbank Accession No. ABI83656.1, SEQ ID NO:21) gene fromNocardia), or the gene products of GriC and GriD from Streptomycesgriseus; followed by conversion to N6-acetyl-1,6-diaminohexane by apolypeptide having ω-transaminase activity classified, for example,under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as from a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 8), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:9), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQID NO: 10), an Escherichia coli (see Genbank Accession No. AAA57874.1,SEQ ID NO: 11), or a Vibrio fluvialis (see Genbank Accession No.AEA39183.1, SEQ ID NO: 12); followed by conversion tohexamethylenediamine by a polypeptide having acetylputrescinedeacetylase activity classified, for example, under EC 3.5.1.17 or EC3.5.1.62. See, FIG. 6.

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor adipate semialdehyde by conversion of adipatesemialdehyde to hexanedial by a polypeptide having carboxylate reductaseactivity classified, for example, under EC 1.2.99.6 such as fromSegniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 6)in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:20) genefrom Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ IDNO:21) gene from Nocardia), or the gene products of GriC and GriD fromStreptomyces griseus; followed by conversion to 6-aminohexanal by apolypeptide having ω-transaminase activity classified, for example,under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48 or EC 2.6.1.82;followed by conversion to hexamethylenediamine by a activityω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:7-12. SeeFIG. 6.

In some embodiments, hexamethylenediamine is synthesized from thecentral precursor 1,6-hexanediol by conversion of 1,6-hexanediol to6-hydroxyhexanal by a a polypeptide having alcohol dehydrogenaseactivity classified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product ofYMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E. coli,GenBank Accession No. AAA69178.1) (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1; followed by conversion of6-oxohexanal to 6-aminohexanol by a polypeptide having ω-transaminaseactivity classified, for example, under EC 2.6.1.- such as 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from aChromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ IDNO: 7), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1,SEQ ID NO: 8), a Pseudomonas syringae (see Genbank Accession No.AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides (see GenbankAccession No. ABA81135.1, SEQ ID NO: 10), an Escherichia coli (seeGenbank Accession No. AAA57874.1, SEQ ID NO: 11), or a Vibrio fluvialis(see Genbank Accession No. AEA39183.1, SEQ ID NO: 12); followed byconversion to 6-aminohexanal by a polypeptide having alcoholdehydrogenase activity classified, for example, under EC 1.1.1.- (e.g.,EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the geneproduct of YMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E.coli, GenBank Accession No. AAA69178.1) (Liu et al., Microbiology, 2009,155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172;Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or theprotein having GenBank Accession No. CAA81612.1; followed by conversionto hexamethylenediamine classified, for example, under EC 2.6.1.- suchas 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 suchas from a Chromobacterium violaceum (see Genbank Accession No.AAQ59697.1, SEQ ID NO: 7), a Pseudomonas aeruginosa (see GenbankAccession No. AAG08191.1, SEQ ID NO: 8), a Pseudomonas syringae (seeGenbank Accession No. AAY39893.1, SEQ ID NO: 9), a Rhodobactersphaeroides (see Genbank Accession No. ABA81135.1, SEQ ID NO: 10), anEscherichia coli (see Genbank Accession No. AAA57874.1, SEQ ID NO: 11),or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO:12). See FIG. 6.

Pathways Using 6-Hydroxyhexanoate and Hexanoyl-CoA as Central Precursorto 1,6-Hexanediol

In some embodiments, 1,6 hexanediol is synthesized from the centralprecursor 6-hydroxyhexanoate by conversion of 6-hydroxyhexanoate to6-hydroxyhexanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 such as from Mycobacteriummarinum (Genbank Accession No. ACC40567.1, SEQ ID NO: 2), Mycobacteriumsmegmatis (Genbank Accession No. ABK71854.1, SEQ ID NO: 3), Segniliparusrugosus (Genbank Accession No. EFV11917.1, SEQ ID NO: 4), Mycobacteriummassiliense (Genbank Accession No. EIV11143.1, SEQ ID NO: 5), orSegniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 6)in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:20) genefrom Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ IDNO:21) gene from Nocardia), or the gene products of GriC and GriD fromStreptomyces griseus (Suzuki et al., J. Antibiot., 2007, 60(6),380-387); followed by conversion of 7-hydroxyhexanal to 1,6 hexanediolby a polypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21,or EC 1.1.1.184 such as the gene product of YMR318C (Genbank AccessionNo. CAA90836.1) or YqhD (from E. coli, GenBank Accession No. AAA69178.1)(from E. coli, GenBank Accession No. AAA69178.1) (see, e.g., Liu et al.,Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J.,361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol.,89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1(from Geobacillus stearothermophilus). See, FIG. 8.

In some embodiments, hexanoyl-CoA can be converted to hexanoic acidhexyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.-(84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 25);followed by conversion to hexanoic acid 6-hydroxy hexyl ester and/or6-hydroxyhexanoic acid 6-hydroxyhexyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.-(1,3). For example, a polypeptide having monooxygenase activitycan be, for example, from the CYP153A family, the CYP52A3 (GenbankAccession No: BAA00371.1, SEQ ID NO: 28) family or the alkB family suchas the gene product of alkBGT from Pseudomonas putida (Genbank AccessionNo. CAB51047.1, SEQ ID NO: 27); followed by conversion of hexanoic acid6-hydroxy hexyl ester and/or 6-hydroxyhexanoic acid 6-hydroxyhexyl to1,6-hexanediol using a polypeptide having esterase activity classified,for example, under EC 3.1.1.-(1,6) such as the gene product of EstC (seeGenBank Accession No. AAC60471.2, SEQ ID NO: 26). See FIG. 8.

Cultivation Strategy

In some embodiments, the cultivation strategy entails achieving anaerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrificationcultivation condition. Enzymes characterized in vitro as being oxygensensitive require a micro-aerobic cultivation strategy maintaining avery low dissolved oxygen concentration (See, for example, Chayabatra &Lu-Kwang, Appl. Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson andBouwer, 1997, Journal of Industrial Microbiology and Biotechnology,18(2-3), 116-130).

In some embodiments, the cultivation strategy entails nutrientlimitation such as nitrogen, phosphate or oxygen limitation.

In some embodiments, a final electron acceptor other than oxygen such asnitrates can be utilized.

In some embodiments, a cell retention strategy using, for example,ceramic membranes can be employed to achieve and maintain a high celldensity during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C6 building blocks can derive frombiological or non-biological feedstocks.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90:885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin andPrather, J. Biotechnol., 2009, 139:61-67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Përez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7):2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Weeet al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al.,J. Bioscience and Bioengineering, 1999, 87(5):647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15):5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1):152-156).

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be a bacterium from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C6 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingone or more C6 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more of such steps. Where less than all the steps areincluded in such a method, the first, and in some embodiments the only,step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host. This documentprovides host cells of any of the genera and species listed andgenetically engineered to express one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms ofany of the enzymes recited in the document. Thus, for example, the hostcells can contain exogenous nucleic acids encoding enzymes catalyzingone or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class. In addition,enzymes in a pathway that require a particular co-factor can be replacedwith an enzyme that has similar or identical activity and specificityfor a different co-factor. For example, one or more steps in a pathwaythat use an enzyme with specificity for NADH can be replaced with anenzyme having similar or identical activity and specificity for NADPH.Similarly, one or more steps in a pathway that use an enzyme withspecificity for NADPH can be replaced with an enzyme having similar oridentical activity and specificity for NADH.

In some embodiments, the enzymes in the pathways outlined herein are theresult of enzyme engineering via non-direct or rational enzyme designapproaches with aims of improving activity, improving specificity,reducing feedback inhibition, reducing repression, improving enzymesolubility, changing stereo-specificity, or changing co-factorspecificity.

In some embodiments, the enzymes in the pathways outlined here can begene dosed, i.e., overexpressed, into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C6 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNAi interference.

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C6 building block.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C6 building block can be improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's endogenous biochemicalnetwork can be attenuated or augmented to (1) ensure the intracellularavailability of acetyl-CoA, (2) create an NADH or NADPH imbalance thatmay be balanced via the formation of one or more C6 building blocks, (3)prevent degradation of central metabolites, central precursors leadingto and including one or more C6 building blocks and/or (4) ensureefficient efflux from the cell.

In some embodiments requiring intracellular availability of acetyl-CoAfor C6 building block synthesis, endogenous enzymes catalyzing thehydrolysis of propionoyl-CoA and acetyl-CoA such as short-chain lengthpolypeptides having thioesterase activity can be attenuated in the hostorganism.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C6 building block synthesis, an endogenous polypeptidehaving phosphotransacetylase activity generating acetate such as pta canbe attenuated (Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for C6 building block synthesis, an endogenous gene in anacetate synthesis pathway encoding a polypeptide having acetate kinaseactivity, such as ack, can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C6 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of pyruvate to lactatesuch as a polypeptide having lactate dehydrogenase activity encoded byldhA can be attenuated (Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C6 building block synthesis, endogenous genesencoding enzymes, such as polypeptide having menaquinol-fumarateoxidoreductase activity, that catalyze the degradation ofphophoenolpyruvate to succinate such as frdBC can be attenuated (see,e.g., Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C6 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of acetyl-CoA toethanol such as the polypeptide having alcohol dehydrogenase activityencoded by adhE can be attenuated (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH co-factor for C6building block synthesis, a recombinant polypeptide having formatedehydrogenase activity gene can be overexpressed in the host organism(Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH or NADPHco-factor for C6 building block synthesis, a polypeptide havingtranshydrogenase activity dissipating the cofactor imbalance can beattenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the degradation of pyruvate to ethanol such as polypeptidehaving pyruvate decarboxylase activity can be attenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the generation of isobutanol such as a polypeptide having2-oxoacid decarboxylase activity can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C6 building block synthesis, a recombinant polypeptidehaving acetyl-CoA synthetase activity such as the gene product of acscan be overexpressed in the microorganism (Satoh et al., J. Bioscienceand Bioengineering, 2003, 95(4):335-341).

In some embodiments, carbon flux can be directed into the butosephosphate cycle to increase the supply of NADPH by attenuating anendogenous polypeptide having glucose-6-phosphate isomerase activity (EC5.3.1.9).

In some embodiments, carbon flux can be redirected into the butosephosphate cycle to increase the supply of NADPH by overexpression apolypeptide having 6-phosphogluconate dehydrogenase activity and/or apolypeptide having transketolase activity (Lee et al., 2003,Biotechnology Progress, 19(5), 1444-1449).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a gene such as UdhA encoding apolypeptide having puridine nucleotide transhydrogenase activity can beoverexpressed in the host organisms (Brigham et al., Advanced Biofuelsand Bioproducts, 2012, Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 Building Block, a recombinant polypeptide havingglyceraldehyde-3-phosphate-dehydrogenase activity gene such as GapN canbe overexpressed in the host organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant malic enzyme genesuch as macA or maeB can be overexpressed in the host organisms (Brighamet al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant polypeptide havingglucose-6-phosphate dehydrogenase activity gene such as zwf can beoverexpressed in the host organisms (Lim et al., J. Bioscience andBioengineering, 2002, 93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant polypeptide havingfructose 1,6 diphosphatase activity such as fbp can be overexpressed inthe host organisms (Becker et al., J. Biotechnol., 2007, 132:99-109).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, endogenous polypeptide havingtriose phosphate isomerase activity (EC 5.3.1.1) can be attenuated.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C6 building block, a recombinant polypeptide havingglucose dehydrogenase activity such as the gene product of gdh can beoverexpressed in the host organism (Satoh et al., J. Bioscience andBioengineering, 2003, 95(4):335-341).

In some embodiments, endogenous enzymes facilitating the conversion ofNADPH to NADH can be attenuated, such as the NADH generation cycle thatmay be generated via inter-conversion of polypeptides having glutamatedehydrogenase activity classified under EC 1.4.1.2 (NADH-specific) andEC 1.4.1.4 (NADPH-specific).

In some embodiments, an endogenous polypeptide having glutamatedehydrogenase activity (EC 1.4.1.3) that utilizes both NADH and NADPH asco-factors can be attenuated.

In some embodiments, a membrane-bound polypeptide having cytochrome P450activity such as CYP4F3B can be solubilized by only expressing thecytosolic domain and not the N-terminal region that anchors the P450 tothe endoplasmic reticulum (see, for example, Scheller et al., J. Biol.Chem., 1994, 269(17):12779-12783).

In some embodiments, a membrane-bound polypeptide having enoyl-CoAreductase activity can be solubilized via expression as a fusion proteinto a small soluble protein such as a maltose binding protein (Gloerichet al., FEBS Letters, 2006, 580, 2092-2096).

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, the endogenous polypeptides havingpolyhydroxyalkanoate synthase activity can be attenuated in the hoststrain.

In some embodiments requiring the intracellular availability ofbutanoyl-CoA for C6 building block synthesis, a recombinant polypeptidehaving propionyl-CoA synthetase activity such as the gene product ofPrpE-RS can be overexpressed in the microorganism (Rajashekhara &Watanabe, FEBS Letters, 2004, 556:143-147).

In some embodiments, a polypeptide having L-alanine dehydrogenaseactivity can be overexpressed in the host to regenerate L-alanine frompyruvate as an amino donor for ω-transaminase reactions.

In some embodiments, a polypeptide having L-glutamate dehydrogenaseactivity, a polypeptide having L-glutamine synthetase activity, or apolypeptide having glutamate synthase activity can be overexpressed inthe host to regenerate L-glutamate from 2-oxoglutarate as an amino donorfor ω-transaminase reactions.

In some embodiments, enzymes such as polypeptide having pimeloyl-CoAdehydrogenase activity classified under, EC 1.3.1.62; a polypeptidehaving acyl-CoA dehydrogenase activity classified, for example, under EC1.3.8.7 or EC 1.3.8.1; and/or a polypeptide having glutaryl-CoAdehydrogenase activity classified, for example, under EC 1.3.8.6 thatdegrade central metabolites and central precursors leading to andincluding C6 building blocks can be attenuated.

In some embodiments, endogenous enzymes activating C6 building blocksvia Coenzyme A esterification such as polypeptide having CoA-ligasesactivity (e.g., a pimeloyl-CoA synthetase) classified under, forexample, EC 6.2.1.14 can be attenuated.

In some embodiments, the efflux of a C6 building block across the cellmembrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C6 building block.

The efflux of hexamethylenediamine can be enhanced or amplified byoverexpressing broad substrate range multidrug transporters such as Bltfrom Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem.,272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins &Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499), NorA fromStaphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother,38(6), 1345-1355), or Bmr from Bacillus subtilis (Neyfakh, 1992,Antimicrob Agents Chemother, 36(2), 484-485).

The efflux of 6-aminohexanoate and hexamethylenediamine can be enhancedor amplified by overexpressing the solute transporters such as the lysEtransporter from Corynebacterium glutamicum (Bellmann et al., 2001,Microbiology, 147, 1765-1774).

The efflux of adipic acid can be enhanced or amplified by overexpressinga dicarboxylate transporter such as the SucE transporter fromCorynebacterium glutamicum (Huhn et al., Appl. Microbiol. & Biotech.,89(2), 327-335).

Producing C6 Building Blocks Using a Recombinant Host

Typically, one or more C6 building blocks can be produced by providing ahost microorganism and culturing the provided microorganism with aculture medium containing a suitable carbon source as described above.In general, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C6 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C6 building block. Once produced, any method can be usedto isolate C6 building blocks. For example, C6 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of adipic acid and 6-aminohexanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case ofhexamethylenediamine and 1,6-hexanediol, distillation may be employed toachieve the desired product purity.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Enzyme Activity of ω-Transaminase Using AdipateSemialdehyde as Substrate and Forming 6-Aminohexanoate

A nucleotide sequence encoding a His-tag was added to the genes fromChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, and Vibrio fluvialis encoding theω-transaminases of SEQ ID NOs: 7, 8, 9, 10 and 12, respectively (seeFIG. 9) such that N-terminal HIS tagged w-transaminases could beproduced. Each of the resulting modified genes was cloned into a pET21aexpression vector under control of the T7 promoter and each expressionvector was transformed into a BL21[DE3] E. coli host. The resultingrecombinant E. coli strains were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 6-aminohexanoateto adipate semialdehyde) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 6-aminohexanoate, 10mM pyruvate and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 6-aminohexanoate and incubated at 25° C. for 24 h,with shaking at 250 rpm. The formation of L-alanine from pyruvate wasquantified via RP-HPLC.

Each enzyme only control without 6-aminoheptanoate demonstrated low baseline conversion of pyruvate to L-alanine See FIG. 16. The gene productof SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 12 accepted6-aminohexanote as substrate as confirmed against the empty vectorcontrol. See FIG. 17.

Enzyme activity in the forward direction (i.e., adipate semialdehyde to6-aminohexanoate) was confirmed for the transaminases of SEQ ID NO 7,SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 12. Enzyme activityassays were performed in a buffer composed of a final concentration of50 mM HEPES buffer (pH=7.5), 10 mM adipate semialdehyde, 10 mM L-alanineand 100 μM pyridoxyl 5′ phosphate. Each enzyme activity assay reactionwas initiated by adding a cell free extract of the ω-transaminase geneproduct or the empty vector control to the assay buffer containing theadipate semialdehyde and incubated at 25° C. for 4 h, with shaking at250 rpm. The formation of pyruvate was quantified via RP-HPLC.

The gene product of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10and SEQ ID NO 12 accepted adipate semialdehyde as substrate as confirmedagainst the empty vector control. See FIG. 18. The reversibility of theω-transaminase activity was confirmed, demonstrating that theω-transaminases of SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10and SEQ ID NO 12 accepted adipate semialdehyde as substrate andsynthesized 6-aminohexanoate as a reaction product.

Example 2 Enzyme Activity of Carboxylate Reductase Using Adipate asSubstrate and Forming Adipate Semialdehyde

A sequence encoding a HIS-tag was added to the genes from Segniliparusrugosus and Segniliparus rotundus that encode the carboxylate reductasesof SEQ ID NOs: 4 and 6, respectively (see FIG. 9), such that N-terminalHIS tagged carboxylate reductases could be produced. Each of themodified genes was cloned into a pET Duet expression vector along with asfp gene encoding a HIS-tagged phosphopantetheine transferase fromBacillus subtilis, both under the T7 promoter. Each expression vectorwas transformed into a BL21[DE3] E. coli host and the resultingrecombinant E. coli strains were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication,and the cell debris was separated from the supernatant viacentrifugation. The carboxylate reductases and phosphopantetheinetransferases were purified from the supernatant using Ni-affinitychromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), andconcentrated via ultrafiltration.

Enzyme activity assays (i.e., from adipate to adipate semialdehyde) wereperformed in triplicate in a buffer composed of a final concentration of50 mM HEPES buffer (pH=7.5), 2 mM adipate, 10 mM MgCl₂, 1 mM ATP and 1mM NADPH. Each enzyme activity assay reaction was initiated by addingpurified carboxylate reductase and phosphopantetheine transferase geneproducts or the empty vector control to the assay buffer containing theadipate and then incubated at room temperature for 20 min. Theconsumption of NADPH was monitored by absorbance at 340 nm. Each enzymeonly control without adipate demonstrated low base line consumption ofNADPH. See FIG. 11.

The gene products of SEQ ID NO 4 and SEQ ID NO 6, enhanced by the geneproduct of sfp, accepted adipate as substrate, as confirmed against theempty vector control (see FIG. 12), and synthesized adipatesemialdehyde.

Example 3 Enzyme Activity of Carboxylate Reductase Using6-Hydroxyhexanoate as Substrate and Forming 6-Hydroxyhexanal

A sequence encoding a His-tag was added to the genes from Mycobacteriummarinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacteriummassiliense, and Segniliparus rotundus that encode the carboxylatereductases of SEQ ID NOs: 2-6, respectively (see FIG. 9) such thatN-terminal HIS tagged carboxylate reductases could be produced. Each ofthe modified genes was cloned into a pET Duet expression vectoralongside a sfp gene encoding a His-tagged phosphopantetheinetransferase from Bacillus subtilis, both under control of the T7promoter. Each expression vector was transformed into a BL21[DE3] E.coli host along with the expression vectors from Example 1. Eachresulting recombinant E. coli strain was cultivated at 37° C. in a 250mL shake flask culture containing 50 mL LB media and antibioticselection pressure, with shaking at 230 rpm. Each culture was inducedovernight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 6-hydroxyhexanoate to 6-hydroxyhexanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 6-hydroxyhexanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 6-hydroxyhexanoate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without6-hydroxyhexanoate demonstrated low base line consumption of NADPH. SeeFIG. 11.

The gene products of SEQ ID NO 2-6, enhanced by the gene product of sfp,accepted 6-hydroxyhexanoate as substrate as confirmed against the emptyvector control (see FIG. 13), and synthesized 6-hydroxyhexanal.

Example 4 Enzyme Activity of ω-Transaminase for 6-Aminohexanol, Forming6-Oxohexanol

A sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the ω-transaminases of SEQ ID NOs: 7-12, respectively (see FIG.9) such that N-terminal HIS tagged ω-transaminases could be produced.The modified genes were cloned into a pET21a expression vector under theT7 promoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 6-aminohexanol to6-oxohexanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 6-aminohexanol, 10mM pyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 6-aminohexanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 6-aminohexanol had low base lineconversion of pyruvate to L-alanine See FIG. 16.

The gene products of SEQ ID NOs: 7-12 accepted 6-aminohexanol assubstrate as confirmed against the empty vector control (see FIG. 21)and synthesized 6-oxohexanol as reaction product. Given thereversibility of the ω-transaminase activity (see Example 2), it can beconcluded that the gene products of SEQ ID NOs: 7-12 accept6-aminohexanol as substrate and form 6-oxohexanol.

Example 5 Enzyme Activity of ω-Transaminase Using Hexamethylenediamineas Substrate and Forming 6-Aminohexanal

A sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the ω-transaminases of SEQ ID NOs: 7-12, respectively (see FIG.9) such that N-terminal HIS tagged ω-transaminases could be produced.The modified genes were cloned into a pET21a expression vector under theT7 promoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e.,hexamethylenediamine to 6-aminohexanal) were performed in a buffercomposed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mMhexamethylenediamine, 10 mM pyruvate, and 100 μM pyridoxyl 5′ phosphate.Each enzyme activity assay reaction was initiated by adding cell freeextract of the ω-transaminase gene product or the empty vector controlto the assay buffer containing the hexamethylenediamine and thenincubated at 25° C. for 4 h, with shaking at 250 rpm. The formation ofL-alanine was quantified via RP-HPLC.

Each enzyme only control without hexamethylenediamine had low base lineconversion of pyruvate to L-alanine See FIG. 16.

The gene products of SEQ ID NOs: 7-12 accepted hexamethylenediamine assubstrate as confirmed against the empty vector control (see FIG. 19)and synthesized 6-aminohexanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 2), it can beconcluded that the gene products of SEQ ID NOs: 7-12 accept6-aminohexanal as substrate and form hexamethylenediamine.

Example 6 Enzyme Activity of Carboxylate Reductase forN6-Acetyl-6-Aminohexanoate, Forming N6-Acetyl-6-Aminohexanal

The activity of each of the N-terminal His-tagged carboxylate reductasesof SEQ ID NOs: 4-6 (see Examples 2 and 3, and FIG. 9) for convertingN6-acetyl-6-aminohexanoate to N6-acetyl-6-aminohexanal was assayed intriplicate in a buffer composed of a final concentration of 50 mM HEPESbuffer (pH=7.5), 2 mM N6-acetyl-6-aminohexanoate, 10 mM MgCl₂, 1 mM ATP,and 1 mM NADPH. The assays were initiated by adding purified carboxylatereductase and phosphopantetheine transferase or the empty vector controlto the assay buffer containing the N6-acetyl-6-aminohexanoate thenincubated at room temperature for 20 min. The consumption of NADPH wasmonitored by absorbance at 340 nm. Each enzyme only control withoutN6-acetyl-6-aminohexanoate demonstrated low base line consumption ofNADPH. See FIG. 11.

The gene products of SEQ ID NO 4-6, enhanced by the gene product of sfp,accepted N6-acetyl-6-aminohexanoate as substrate as confirmed againstthe empty vector control (see FIG. 14), and synthesizedN6-acetyl-6-aminohexanal.

Example 7 Enzyme Activity of ω-Transaminase UsingN6-Acetyl-1,6-Diaminohexane, and Forming N6-Acetyl-6-Aminohexanal

The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs:7-12 (see Example 4, and FIG. 9) for convertingN6-acetyl-1,6-diaminohexane to N6-acetyl-6-aminohexanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM N6-acetyl-1,6-diaminohexane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase or theempty vector control to the assay buffer containing theN6-acetyl-1,6-diaminohexane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N6-acetyl-1,6-diaminohexanedemonstrated low base line conversion of pyruvate to L-alanine See FIG.14.

The gene product of SEQ ID NOs: 7-12 acceptedN6-acetyl-1,6-diaminohexane as substrate as confirmed against the emptyvector control (see FIG. 20) and synthesized N6-acetyl-6-aminohexanal asreaction product.

Given the reversibility of the ω-transaminase activity (see Example 2),the gene products of SEQ ID NOs: 7-12 accept N6-acetyl-6-aminohexanal assubstrate forming N6-acetyl-1,6-diaminohexane.

Example 8 Enzyme Activity of Carboxylate Reductase Using AdipateSemialdehyde as Substrate and Forming Hexanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO 6 (seeExample 2, Example 3 and FIG. 9) was assayed using adipate semialdehydeas substrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM adipate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the adipate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout adipate semialdehyde demonstrated low base line consumption ofNADPH. See FIG. 11.

The gene product of SEQ ID NO: 6, enhanced by the gene product of sfp,accepted adipate semialdehyde as substrate as confirmed against theempty vector control (see FIG. 15) and synthesized hexanedial.

Example 9 Enzyme Activity of CYP153 Monooxygenase Using Hexanoate asSubstrate in Forming 6-Hydroxyhexanoate

A sequence encoding a HIS tag was added to the Polaromonas sp. JS666,Mycobacterium sp. HXN-1500 and Mycobacterium austroafricanum genesrespectively encoding (1) the monooxygenases (SEQ ID NOs: 13-15), (2)the associated ferredoxin reductase partner (SEQ ID NOs: 16-17) and thespecie's ferredoxin (SEQ ID NOs: 18-19). For the Mycobacteriumaustroafricanum monooxygenase, Mycobacterium sp. HXN-1500 oxidoreductaseand ferredoxin partners were used. The three modified protein partnerswere cloned into a pgBlue expression vector under a hybrid pTacpromoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 500 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure. Each culture was induced for 24 h at 28°C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and the cells made permeableusing Y-Per™ solution (ThermoScientific, Rockford, Ill.) at roomtemperature for 20 min. The permeabilized cells were held at 0° C. inthe Y-Per™ solution.

Enzyme activity assays were performed in a buffer composed of a finalconcentration of 25 mM potassium phosphate buffer (pH=7.8), 1.7 mMMgSO₄, 2.5 mM NADPH and 30 mM hexanoate. Each enzyme activity assayreaction was initiated by adding a fixed mass of wet cell weight ofpermeabilized cells suspended in the Y-Per™ solution to the assay buffercontaining the heptanoate and then incubated at 28° C. for 24 h, withshaking at 1400 rpm in a heating block shaker. The formation of7-hydroxyheptanoate was quantified via LC-MS.

The monooxygenase gene products of SEQ ID NO 13-15 along with reductaseand ferredoxin partners, accepted hexanoate as substrate as confirmedagainst the empty vector control (see FIG. 22) and synthesized6-hydroxyhexanoate as reaction product.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of producing a terminal hydroxyl (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester in a recombinant host, said methodcomprising: a) enzymatically converting a C₄₋₉ carboxylic acid to a(C₃₋₈ alkyl)-C(═O)OCH₃ ester using a polypeptide having fatty acidO-methyltransferase activity classified under EC 2.1.1.15; and b)enzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to a terminalhydroxyl (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester using a polypeptide havingmonooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.-,wherein the C₄₋₉ carboxylic acid is enzymatically produced from a C₄₋₉alkanoyl-CoA using: a polypeptide having thioesterase activityclassified under EC 3.1.2.-; or a polypeptide having butanaldehydrogenase activity classified under EC 1.2.1.10 or EC 1.2.1.57 and apolypeptide having aldehyde dehydrogenase activity classified under EC1.2.1.3 or EC 1.2.1.4.
 2. The method of claim 1, said method furthercomprising enzymatically converting the terminal hydroxyl (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester to a terminal hydroxyl C₄₋₉hydroxyalkanoate using a polypeptide having demethylase activityclassified under EC 2.1.1.- and/or a polypeptide having esteraseactivity classified under EC 3.1.1.-.
 3. The method of claim 1, whereinC₄₋₉ carboxylic acid is hexanoate, and is enzymatically converted tohexanoate methyl ester; and the hexanoate methyl ester is enzymaticallyconverted to 6-hydroxyhexanoate methyl ester.
 4. The method of claim 1,wherein the C₄₋₉ alkanoyl-CoA is hexanoyl-CoA, and hexanoate isenzymatically produced from hexanoyl-CoA.
 5. The method of claim 3, saidmethod further comprising enzymatically converting 6-hydroxyhexanoatemethyl ester to 6-hydroxyhexanoate using a polypeptide havingdemethylase activity classified under EC 2.1.1.- or a polypeptide havingesterase activity classified under EC 3.1.1.-.
 6. The method of claim 4,wherein hexanoyl-CoA is produced from acetyl-CoA via two cycles ofCoA-dependent carbon chain elongation, wherein each of said two cyclesof CoA-dependent carbon chain elongation comprises using (i) apolypeptide having β-ketothiolase activity classified under EC 2.3.1.9,EC 2.3.1.16, or EC 2.3.1.174 or a polypeptide having acetyl-CoAcarboxylase activity classified under EC 6.4.1.2 and a polypeptidehaving acetoacetyl-CoA synthase activity classified under EC 2.3.1.194,(ii) a polypeptide having 3-hydroxyacyl-CoA dehydrogenase activityclassified under EC 1.1.1.35, EC 1.1.1.36, or EC 1.1.1.157 or apolypeptide having 3-oxoacyl-CoA reductase activity classified under EC1.1.1.100, (iv) a polypeptide having enoyl-CoA hydratase activityclassified under EC 4.2.1.17 or EC 4.2.1.119, and (v) a polypeptidehaving trans-2-enoyl-CoA reductase activity classified under EC 1.3.1.8,EC 1.3.1.38, or EC 1.3.1.44 to form hexanoyl-CoA from acetyl-CoA.
 7. Amethod of enzymatically producing 6-hydroxyhexanoate, the methodcomprising: a) enzymatically converting hexanoate to hexanoate methylester using a polypeptide having fatty acid O-methyltransferase activityclassified under EC 2.1.1.15; b) enzymatically converting hexanoatemethyl ester to 6-hydroxyhexanoate methyl ester using a polypeptidehaving monooxygenase activity classified under EC 1.14.14.- or EC1.14.15.-; and c) enzymatically converting 6-hydroxyhexanoate methylester to 6-hydroxyhexanoate using a polypeptide having demethylaseactivity classified under EC 2.1.1.- or a polypeptide having esteraseactivity classified under EC 3.1.1.-.
 8. The method of claim 1, whereinsaid recombinant host is subjected to a cultivation strategy underaerobic, anaerobic or, micro-aerobic cultivation conditions.
 9. Themethod of claim 8, wherein said recombinant host is cultured underconditions of nutrient limitation.
 10. The method of claim 8, whereinsaid recombinant host is retained using a ceramic membrane to maintain ahigh cell density during fermentation.
 11. The method of claim 8,wherein the principal carbon source fed to the recombinant host derivesfrom a biological feedstock.
 12. The method of claim 11, wherein thebiological feedstock is, or derives from, monosaccharides,disaccharides, lignocellulose, hemicellulose, cellulose, lignin,levulinic acid, formic acid, triglycerides, glycerol, fatty acids,agricultural waste, condensed distillers' solubles, or municipal waste.13. The method of claim 8, wherein the principal carbon source fed tothe recombinant host derives from a non-biological feedstock.
 14. Themethod of claim 13, wherein the non-biological feedstock is, or derivesfrom, natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) caustic wash waste stream from cyclohexaneoxidation processes, or terephthalic acid/isophthalic acid mixture wastestreams.
 15. The method of claim 9, wherein the recombinant host is aprokaryote selected from Escherichia, Clostridia, Corynebacteria,Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus,and Rhodococcus, or a eukaryote selected from Aspergillus,Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, andKluveromyces.
 16. The method of claim 3, wherein the polypeptide havingfatty acid O-methyltransferase activity classified under EC 2.1.1.15 hasat least 85% sequence identity to an amino acid sequence set forth inSEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24; and the polypeptide havingmonooxygenase activity classified under EC 1.14.14.- or EC 1.14.15.- hasat least 85% sequence identity to an amino acid sequence set forth inSEQ ID NO: 13-15, SEQ ID NO:27 or SEQ ID NO:28.
 17. The method of claim4, wherein the polypeptide having thioesterase activity classified underEC 3.1.2.- has at least 85% sequence identity to the amino acid sequenceset forth in SEQ ID NO:1, SEQ ID NO:32, or SEQ ID NO:33.
 18. The methodof claim 5, wherein the polypeptide having demethylase activityclassified under EC 2.1.1.- has at least 85% sequence identity to theamino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 and thepolypeptide having esterase activity classified under EC 3.1.1.- has atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:
 26. 19. The method of claim 8, wherein said recombinant host iscultured under conditions of phosphate, nitrogen, or oxygen limitation.